Modified halogenated polymer surfaces

ABSTRACT

Disclosed is a method of preparing a modified halogenated polymer surface, comprising the steps of (a) activating the surface by modification with a polymerisation initiator by (a 1 ) reacting the halogenated polymer surface with sodium azide and subsequent (a 2 ) 1,3 dipolar cycloaddition with an alkine-functionalized initiator; or (a 3 ) reacting the halogenated polymer surface with mercapto-functionalized initiators; and (b) reacting the activated surface obtained in steps (a 1 )/(a 2 ) or (a 3 ) with polymerizable monomeric units A and/or B. The modified halogenated polymer substrates according to the invention exhibit outstanding properties.

The present invention relates to a method of preparing modifiedhalogenated polymer surfaces and the surface-modified halogenatedpolymer substrates prepared from halogenated polymers according to thismethod.

The surface properties of polymeric materials are important to many oftheir applications.

Due to the steadily growing importance of microtechniques in a widevariety of scientific applications, the development of systems whichallow the interaction of molecules with surfaces remains a criticalissue. Such interactions include the possibility of removing specificmolecules from a sample, e.g. to facilitate their analysis/detection,but also of presenting molecules on a surface, thus allowing subsequentreactions to take place. These principles for the immobilization ofmolecules can be applied in sensor or chromatographic systems or for theprovision of modified surfaces in general.

In recent years there have been numerous approaches to fabricate sensorchips which are based on self-assembled monolayers (SAM's) ofbifunctional molecules which directly or indirectly couple samplemolecules to the sensor surface. Typically, these bifunctional moleculescarry a silane or thiol/disulfide moiety in order to achieve a bond withan inorganic surface and an additional functional group (e.g. amino orepoxide groups) which interact with sample molecules, often contained inbiological samples in the form of an oligonucleotide, a protein or apolysaccharide etc.

A desired polymer surface can often not be obtained from the materialitself but with modification.

Modifications of polymer surfaces can be obtained both by variousphysical and chemical processes.

It is well known prior art that PVC films can be modified andfunctionalized at the surface with small molecules such as thiolates orazide via nucleophilic substitution of chlorine atoms by wet-chemicaltreatments using mixtures of solvents and non-solvents for the polymeror by using a phase transfer catalyst like nBu₄NBr in aqueous solutions(J. Sacristán, C. Mijangos, H. Reinecke, Polymer 2000, 41 5577-5582; A.Jayakrishnan, M. C. Sunny, Polymer 1996, 37, 5213-5218).

Methods of modifying plasticized PVC films by wet-chemical modificationmethods are disclosed in J. Sacristán, C. Mijangos, H. Reinecke, Polymer2000, 41, 5577-5582; J. Reyes-Labarta, M. Herrero, P. Tiemblo, C.Mijangos, H. Reinecke, Polymer 2003, 44, 2263-2269; M. Herrero, R.Navarro, N. Garcia, C. Mijangos, H. Reinecke, Langmuir, 2005, 21,4425-4430.

The described modified PVC films do not encompass PVC films having anoligomeric or polymeric unit bond to the PVC film.

Living polymerization systems have been developed which allow for thecontrol of molecular weight, end group functionality, andarchitecture.[Webster, O. Science, 1991, 251 887].

Most notably, these systems involve ionic polymerization. As thesepolymerization systems are ionic in nature, the reaction conditionsrequired to successfully carry out the polymerization include thecomplete exclusion of water from the reaction medium. Another problemwith ionic living polymerizations is that one is restricted in thenumber of monomers which can be successfully polymerized. Also, due tothe high chemoselectivity of the propagating ionic centers, it is verydifficult, if not impossible, to obtain random copolymers of two or moremonomers; block copolymers are generally formed.

Radical polymerization is one of the most widely used methods forpreparing high polymer from a wide range of vinyl monomers. Althoughradical polymerization of vinyl monomers is very effective, it does notallow for the direct control of molecular weight(DP_(n)≠Δ[Monomer]/[Initiator]_(o)), control of chain endfunctionalities or for the control of the chain architecture, e.g.,linear vs. branched or graft polymers. In the past five years, muchinterest has been focused on developing a polymerization system which isradical in nature but at the same time allows for the high degree ofcontrol found in the ionic living systems.

A polymerization system has been previously disclosed that does providefor the control of molecular weight, end groups, and chain architecture,and that was radical in nature, (K. Matyjaszewski, J. -S. Wang,Macromolecules 1995, 28, 7901-7910; K. Matyjaszewski, T. Patten, J. Xia,T. Abernathy, Science 1996, 272, 866-868; U.S. Pat. No. 5,763,548; U.S.Pat. No. 5,807,937; U.S. Pat. No. 5,789,487) the contents of which arehereby incorporated by reference. This process has been termed atomtransfer radical polymerization (ATRP). ATRP employs the reversibleactivation and deactivation of a compound containing a radicallytransferable atom or group to form a propagating radical (R•) by a redoxreaction between the radical and a transition metal complex (M_(t)^(n-1)) with a radically transferable group (X).

Controlled polymerization is initiated by use, or formation, of amolecule containing a radically transferable atom or group. Previouswork has concentrated on the use of an alkyl halide adjacent to a groupwhich can stabilize the formed radical. Other initiators may containinorganic/pseudo halogen groups which can also participate in atomtransfer, such as nitrogen, oxygen, phosphorous, sulfur, tin, etc.

The most important aspect of the reaction outlined in Scheme 1 is theestablishment of an equilibrium between the active radicals and thedormant species, R—X (dormant polymer chains=P_(n)-X). Understanding andcontrolling the balance of this equilibrium is very important incontrolling the radical polymerization. If the equilibrium is shiftedtoo far towards the dormant species, then there would be nopolymerization. However, if the equilibrium is shifted too far towardsthe active radical, too many radicals are formed resulting inundesirable bimolecular termination between radicals. This would resultin a polymerization that is not controlled. An example of this type ofirreversible redox initiation is the use of peroxides in the presence ofiron (II). By obtaining an equilibrium which maintains a low, but nearlyconstant concentration of radicals, bimolecular termination betweengrowing radicals can be suppressed, one obtains high polymer.

Surprisingly it has been found that modified halogenated polymersurfaces can be obtained by covalent binding of a radical initiator onthe surface of the halogenated polymer and subsequent grafting polymersof defined composition on this modified halogenated polymer surface in acontrolled polymerization reaction.

The halogenated polymer surface modified in this manner exhibits newproperties.

Therefore, the present invention relates to a method of preparing amodified halogenated polymer surface, comprising the steps of

(a) activating the surface by modification with a polymerisationinitiator by

-   -   (a₁) reacting the halogenated polymer surface with sodium azide        and subsequent    -   (a₂) 1,3 dipolar cycloaddition with an alkine-functionalized        initiator; or alternatively    -   (a₃) reacting the halogenated polymer surface with        mercapto-functionalized initiators; and

(b) reacting this activated surface obtained in steps (a₁)/(a₂) or (a₃)with polymerizable monomeric units A and/or B.

In the first reaction step (a₁) the halogenated polymer substrate istreated with sodium azide in a manner known per se as for exampledisclosed by A. Jayakrishnan, M. C. Sunny, Polymer 1996, 37, 5213-5218.

In this reaction step the azide group will be covalently bonded on thesurface of the halogenated polymer.

This reaction is preferably carried out in a 1% to 25% aqueous solutionof sodium azide at a temperature from 20° C. to 100° C., preferably from60° C. to 90° C.

The reaction time is from 0.1 h to 2 h, preferably 1 h to 4 h.

The reaction is preferably carried out in the presence of a phasetransfer catalyst, more preferably in the presence of n-tetrabutylammonium bromide.

The activation of the surface can be controlled by IR spectroscopy dueto the strong IR activity of the azide.

The degree of modification of the halogenated polymer substrate dependson reaction parameters like reaction time, temperature, solvents and theconcentration of the reagents.

The reaction (a₁) comprises the steps of interaction of the surface ofthe polymer substrate with the reaction medium (a_(1a)), whichcontemplates the diffusion of the solvent into the upper part of thesurface, the second step is the transport of the modification agent tothe functional group of the polymer (a_(1b)), and the third step is thereaction itself (a_(1c)).

The reaction step (a₁) can be illustrated by the following reactionscheme:

Reaction step (a₂) represents a copper-catalyzed 1,3 dipolarcycloaddition with an alkine-functionalized initiator. This reaction isknown as Huisgen- or click-reaction.

The reaction step (a₂) can be illustrated by the following reactionscheme:

In this reaction step a suitable initiator is bonded to the halogenatedpolymer substrate.

This reaction is preferably carried out in a 0.1% to 10% solution of therespective alkine in iso-propanol at a temperature from 20° C. to 100°C., preferably at 50° C. to 80° C.

The reaction time is from 0.1 h to 24 h, preferably 10 h to 16 h.

The reaction is preferably carried out in the presence of a coppercatalyst and a base, more preferably in the presence of Cu[MeCN]₄PF₆ and2,6-lutidine.

The reaction can be controlled by IR spectroscopy due to the strong IRactivity of the carbonyl-moiety.

Examples of halogenated polymers include

Halopolymers include organic polymers which contain halogenated groups,such as chloropolymers, fluoropolymers and fluorochloropolymers.Examples of halopolymers include fluoroalkyl, difluoroalkyl,trifluoroalkyl, fluoroaryl, difluoroaryl, trifluoroaryl, perfluoroalkyl,perfluoroaryl, chloroalkyl, dichloroalkyl, trichloroalkyl, chloroaryl,dichloroaryl, trichloroaryl, perchloroalkyl, perchloroaryl,chlorofluoroalkyl, chlorofluoroaryl, chlorodifluoroalkyl, anddichlorofluoroalkyl groups. Halopolymers also include fluorohydrocarbonpolymers, such as polyvinylidine fluoride (“PVDF”), polyvinylflouride(“PVF”), polychlorotetrafluoroethylene (“PCTFE”),polytetrafluoroethylene (“PTFE”) (including expanded PTFE (“ePTFE”)).Other halopolymers include fluoropolymers perfluorinated resins, such asperfluorinated siloxanes, perfluorinated styrenes, perfluorinatedurethanes, and copolymers containing tetrafluoroethylene and otherperfluorinated oxygen-containing polymers likeperfluoro-2,2-dimethyl-1,3-dioxide (which is sold under the trade nameTEFLON-AF). Still other halopolymers which can be used in the practiceof the present invention include perfluoroalkoxy-substitutedfluoropolymers, such as MFA (available from Ausimont USA (Thoroughfare,N.J.)) or PFA (available from Dupont (Willmington, Del.)),polytetrafluoroethylene-co-hexafluoropropylene (“FEP'”),ethylenechlorotrifluoroethylene copolymer (“ECTFE”), and polyester basedpolymers, examples of which include polyethyleneterphthalates,polycarbonates, and analogs and copolymers thereof.

Halogen-containing polymers comprise polychloroprene, chlorinatedrubbers, chlorinated and brominated copolymer of isobutylene-isoprene(halobutyl rubber), chlorinated or sulfochlorinated polyethylene,copolymers of ethylene and chlorinated ethylene, epichlorohydrin homo-and copolymers, especially polymers of halogen-containing vinylcompounds, for example polyvinyl chloride, polyvinylidene chloride,polyvinyl fluoride, polyvinylidene fluoride, as well as copolymersthereof such as vinyl chloride/vinylidene chloride, vinyl chloride/vinylacetate or vinylidene chloride/vinyl acetate copolymers.

The term “polyvinyl chloride” means compositions whose polymer is avinyl chloride homopolymer. The homopolymer may be chemically modified,for example by chlorination.

They are in particular polymers obtained by copolymerization of vinylchloride with monomers containing an ethylenically polymerizable bond,for instance vinyl acetate, vinylidene chloride; maleic or fumaric acidor esters thereof; olefins such as ethylene, propylene or hexene;acrylic or methacrylic esters; styrene; vinyl ethers such as vinyldodecyl ether.

The compositions according to the invention may also contain mixturesbased on chlorinated polymers containing minor quantities of otherpolymers, such as halogenated polyolefins oracrylonitrile/butadiene/styrene copolymers.

Usually, the copolymers contain at least 50% by weight of vinyl chlorideunits and preferably at least 80% by weight of such units.

In general, any type of polyvinyl chloride is suitable, irrespective ofits method of preparation. Thus, the polymers obtained, for example, byperforming bulk, suspension or emulsion processes may be stabilisedusing the composition according to the invention, irrespective of theintrinsic viscosity of the polymer.

Preferably, the initiator represents the fragment of a polymerizationinitiator capable of initiating polymerization of ethylenicallyunsaturated monomers in the presence of a catalyst which activatescontrolled radical polymerization.

The initiator is preferably selected from the group consisting ofC₁-C₈-alkylhalides, C₆-C₁₅-aralkylhalides, C₂-C₈-haloalkyl esters, arenesulphonyl chlorides, haloalkanenitriles, α-haloacrylates andhalolactones.

Specific initiators are selected from the group consisting ofα,α′-dichloro- or α,α′-dibromoxylene, p-toluenesulfonylchloride (PTS),hexakis-(α-chloro- or α-bromomethyl)-benzene, 1-phenethyl chloride orbromide, methyl or ethyl 2-chloro- or 2-bromopropionate, methyl orethyl-2-bromo- or 2-chlorooisobutyrate, and the corresponding 2-chloro-or 2-bromopropionic acid, 2-chloro- or 2-bromoisobutyric acid, chloro-or bromoacetonitrile, 2-chloro- or 2-bromo-propionitrile,α-bromo-benzacetonitrile, α-bromo-γ-butyrolactone(=2-bromo-dihydro-2(3H)-furanone) and the initiators derived from1,1,1-(tris-hydroxymethyl)propane and pentaerythritol of the formulae ofabove.

ATRP Initiators

Initiators for ATRP can be prepared by a variety of methods. Since allthat is needed for an ATRP initiator is a radically transferable atom orgroup, such as a halogen, standard organic synthetic techniques can beapplied to preparing ATRP initiators. Some general methods for preparingATRP initiators will be described here. In general the initiators canhave the general formula: Y—(X)_(n). wherein Y is the core of themolecule and X is the radically transferable atom or group. The number ncan be any number 1 or higher, depending on the functionality of thecore group Y. For example, when Y is benzyl and X is bromine, with n=1,the resulting compound is benzyl bromide. If Y is a phenyl moeity havinga CH₂ group attached to each carbon of the phenyl ring and X is Br withn=6, the compound is hexa(bromomethyl)benzene, a hexafunctionalinitiator useful for the preparation of six polymer chains from a singleinitiator.

As a first division of the initiator types, there are two classes, smallmolecule and macro-molecule. The small molecule initiators can becommercially available, such as benzylic halides, 2-halopropionates and2-haloisobutyrates, 2-halopropionitriles, α-halomalonates, tosylhalides, carbon tetrahalides, carbon trihalides, etc. Of course, thesefunctional groups can be incorporated into other small molecules. Theincorporation of these functional groups can be done as a singlesubstitution, or the small molecule can have more than one initiatingsite for ATRP. For example, a molecule containing more than one hydroxylgroup can undergo an esterification reaction to generate α-haloesterswhich can initiate ATRP. Of course, other initiator residues can beintroduced as are desired. The small molecules to which the initiatorsare attached can be organic or inorganic based; so long as the initiatordoes not poison the catalyst or adversely interact with the propagatingradical it can be used. Some examples of small molecules that were usedas a foundation for the attachment of initiating sites arepolydimethylsiloxane cubes, cyclotriphosphazene rings,2-tris(hydroxyethyl)ethane, glucose based compounds, etc. Additionally,trichloromethyl isocyanate can be used to attach an initiator residue toany substance containing hydroxy, thiol, amine and/or amide groups.

Macroinitiators can take many different forms, and can be prepared bydifferent methods. The macroinitiators can be soluble polymers,insoluble/crosslinked polymeric supports, surfaces, or solid inorganicsupports. Some general methods for the preparation of themacroinitiators include modification of an existing material,(co)polymerization of an AB* monomer by ATRP/non-ATRP methods, or usinginitiators (for other types of polymerization) that contain an ATRPinitiator residue. Again, modification of macromolecularcompounds/substrates to generate an ATRP initiation site isstraightforward to one skilled in the art of materials/polymermodification. For example, crosslinked polystyrene with halomethylgroups on the phenyl rings (used in solid-phase peptide synthesis),attached functional molecules to silica surfaces, brominated solublepolymers (such as (co)polymers of isoprene, styrene, and othermonomers), or attached small molecules containing ATRP initiators topolymer chains can all be used as macromolecular initiators. If one ormore initiating sites are at the polymer chain ends, then block(co)polymers are prepared; if the initiating sites are dispersed alongthe polymer chain, graft (co)polymers will be formed.

AB* monomers, or any type of monomer that contains an ATRP initiatorresidue, can be (co)polymerized, with or without other monomers, byvirtually any polymerization process, except for ATRP to prepare linearpolymers with pendant B* groups. The only requirement is that the ATRPinitiator residue remains intact during and after the polymerization.This polymer can then be used to initiate ATRP when in the presence of asuitable vinyl monomer and ATRP catalyst. When ATRP is used to(co)polymerize the AB* monomers, (hyper)branched polymers will result.Of course, the macromolecules can also be used to initiate ATRP.

Functionalized initiators for other types of polymerization systems,i.e., conventional free radical, cationic ring opening, etc., can alsobe used. Again, the polymerization mechanism should not involve reactionwith the ATRP initiating site. Also, in order to obtain pure blockcopolymers, each chain of the macroinitiator must be initiated by theoriginal functionalized initiator. Some examples of these type ofinitiators would include functionalized azo compounds and peroxides(radical polymerization), functionalized transfer agents (cationic,anionic, radical polymerization), and 2-bromopropionyl bromide/silvertriflate for the cationic ring opening polymerization oftetrahydrofuran.

The ATRP initiators can be designed to perform a specific function afterbeing used to initiate ATRP reactions. For example, biodegradable(macro)initiators can be used as a method to recycle or degradecopolymers into reusable polymer segments. An example of this would beto use a difunctional biodegradable initiator to prepare a telechelicpolymer. Since telechelic polymers can be used in step-growthpolymerizations, assuming properly functionalized, linear polymers canbe prepared with multiple biodegradable sites along the polymer chains.Under appropriate conditions, i.e., humidity, enzymes, etc., thebiodegradable segments can break down, and the vinyl polymer segmentsrecovered and recycled. Additionally, siloxane containing initiators canbe used to prepare polymer with siloxane end groups/blocks. Thesepolymers can be used in sol-gel processes.

It is also possible to use multifunctional initiators having one or moreinitiation sites for ATRP and one or more initiation sites capable ofinitiating a non-ATRP polymerization. The non-ATRP polymerization caninclude any polymerization mechanism, including, but not limited to,cationic, anionic, free radical, metathesis, ring opening andcoordination polymerizations. Exemplary multifunctional initiatorsinclude, but are not limited to, 2-bromopropionyl bromide (for cationicor ring opening polymerizations and ATRP); halogenated AIBN derivativesor halogenated peroxide derivatives (for free radical and ATRPpolymerizations); and 2-hydroxyethyl 2-bromopropionate (for anionic andATRP polymerizations).

Reverse ATRP is the generation, in situ, of the initiator containing aradically transferable group and a lower oxidation state transitionmetal, by use of a conventional radical initiator and a transition metalin a higher oxidation state associated with a radically transferableligand (X), e.g., Cu (II) Br₂, using the copper halide as a model. Whenthe conventional free radical initiator decomposes, the radical formedmay either begin to propagate or may react directly with theM^(n-1)X_(y)L (as can the propagating chain) to form an alkyl halide andM^(n)X_(y-1)L. After most of the initiator/M^(n-1)X_(y)L is consumed,predominately the alkyl halide and the lower oxidation metal species arepresent; these two can then begin ATRP.

Previously, Cu(II)X₂/bpy and AIBN have been used as a reverse ATRPcatalyst system. (U.S. Pat. No. 5,763,548, K, Matyjaszewski, J. -S.Wang, Macromolecules 1995, 28, 7572-7573) However, molecular weightswere difficult to control and polydispersities were high. Also, theratio of Cu(II) to AIBN was high, 20:1. The present invention providesan improved reverse ATRP process using dNbpy, to solubilize thecatalyst, which leads to a significant improvement in the control of thepolymerization and reduction in the amount of Cu(II) required.

Reverse ATRP can now be successfully used for the “living”polymerization of monomers such as styrene, methyl acrylate, methylmethacrylate, and acrylonitrile. The polymer molecular weights obtainedagree with theory and polydispersities are quite low, M_(w)/M_(n).=1.2.Due to the enhanced solubility of the Cu(II) by using dNbpy, as theligand, the ratio of Cu(II):AIBN can be drastically reduced to a ratioof 1:1. Unlike standard AIBN initiated polymerizations, the reverse ATRPinitiated polymers all have identical 2-cyanopropyl (from decompositionof AIBN) head groups and halogen tail groups which can further beconverted into other functional groups. Additionally, substituents onthe free radical initiator can be used to introduce additionalfunctionality into the molecule.

The radical initiator used in reverse ATRP can be any conventionalradical initiator, including but not limited to, organic peroxides,organic persulfates, inorganic persulfates, peroxydisulfate, azocompounds, peroxycarbonates, perborates, percarbonates, perchlorates,peracids, hydrogen peroxide and mixtures thereof. These initiators canalso optionally contain other functional groups that do not interferewith ATRP.

Alternatively, the activation of the halogenated polymer surface bymodification with a polymerisation initiator can be carried out by athiol-substituted initiator (reaction step (a₃)). In this case thesulphur reacts as a nucleophile and the corresponding initiator can bebonded at the halogenated polymer surface by substitution of the chloroatom.

The reaction step (a₃) can be illustrated by the following reactionscheme:

In the reaction step (b) the polymerizable monomeric units A and B arepreferably copolymerized by atom transfer radical polymerization (ATRP)participating the initiator of the activated surface obtained in steps(a₁)/(a₂) or (a₃).

The ATRP method enables the production of so called “polymer brushes” onthe modified halogenated polymer surface, i.e. covalently bound polymerchains of defined composition with low polydispersity and exclusion fromcross linking. It is to be noted, that the polymer brushes formed in theinvention may also be formed by several other polymerization methods,which are standart in the art, including but not limited to RAFT, NMPand ROMP.

In principal it is possible to carry out the polymerization with themonomeric unit A following the reaction with the monomeric unit B.

It is also possible to carry out the polymerization reaction with amixture of the monomeric units A and B.

The halogenated polymer substrate, for example in form of a film, whichwas modified according to reaction steps (a₁), (a₂) or (a₃) is reactedin a further reaction step (b) with the corresponding monomer undersuitable conditions.

The reaction step (b) can be illustrated by the following reactionscheme:

This reaction is preferably carried out in a 5% to 50% solution of therespective monomer in a mixture of water and an alcohol or in an alcoholat a temperature from 20° C. to 100° C., preferably at 20° C. to 60° C.

The reaction time is from 0.1 h to 24 h, preferably 1 h to 4 h.

The reaction is preferably carried out in the presence of a catalystsystem, more preferably in the presence of CuBr, Cubr₂ and Bipyridin.

Monomers

The monomers useful in the present polymerization processes can be anyradically (co)polymerizable monomers. Within the context of the presentinvention, the phrase “radically (co)polymerizable monomer” indicatesthat the monomer can be either homopolymerized by radical polymerizationor can be radically copolymerized with another monomer, even though themonomer in question cannot itself be radically homopolymerized. Suchmonomers typically include any ethylenically unsaturated monomer,including but not limited to, styrenes, acrylates, methacrylates,acrylamides, acrylonitriles, isobutylene, dienes, vinyl acetate,N-cyclohexyl maleimide, 2-hydroxyethyl acrylates, 2-hydroxyethylmethacrylates, and fluoro-containing vinyl monomers. These monomers canoptionally be substituted by any substituent that does not interferewith the polymerization process, such as alkyl, alkoxy, aryl,heteroaryl, benzyl, vinyl, allyl, hydroxy, epoxy, amide, ethers, esters,ketones, maleimides, succinimides, sulfoxides, glycidyl or silyl.

The polymers may be prepared from a variety of monomers. A particularlyuseful class of water-soluble or water-dispersible monomers featuresacrylamide monomers having the formula:

where R₄ is H or an alkyl group; and R₅ and R₆, independently, areselected from the group consisting of hydrogen, alkyl, substitutedalkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl,heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, aryloxy, and combinationsthereof; R₅ and R₆ may be joined together in a cyclic ring structure,including heterocyclic ring structure, and that may have fused with itanother saturated or aromatic ring. An especially preferred embodimentis where R₅ and R₆, independently, are selected from the groupconsisting of hydroxy-substituted alkyl, polyhydroxy-substituted alkyl,amino-substituted alkyl, polyamino-substituted alkyl andisothiocyanato-substituted alkyl. In preferred embodiments, the polymersinclude the acrylamide-based repeat units derived from monomers such asacrylamide, methacrylamides, N-alkylacrylamide (e.g.,N-methylacrylamide, N-tert-butylacrylamide, and N-n-butylacrylamide),N-alkylmethacrylamide (e.g., N-tert-butylmethacrylamide andN-n-butylmethacrylamide), N,N-dialkylacrylamide (e.g.,N,N-dimethylacrylamide), N-methyl-N-(2-hydroxyethyl)acrylamide,N,N-di-alkylmethacrylamide, N-methylolmethacrylamide,N-ethylolmethacrylamide, N-methylolacrylamide, N-ethylolacrylamide, andcombinations thereof. In another preferred embodiment, the polymersinclude acrylamidic repeat units derived from monomers selected fromN-alkylacrylamide, N-alkylmethacrylamide, N,N-dialkylacrylamide andN,N-dialkylmethacrylamide. Preferred repeat units can be derived,specifically, from acrylamide, methacrylamide, N,N-dimethylacrylamide,and tert-butylacrylamide.

Copolymers can include two or more of the aforementionedacrylamide-based repeat units. Copolymers can also include, for example,one or more of the aforementioned polyacrylamide-based repeat units incombination with one or more other repeat units.

Generally speaking, in some embodiments of the present invention themonomer may be represented by the formula

wherein

P is a functional group that polymerizes in the presence of freeradicals (e.g., a carbon-carbon double bond), and E is a group that canreact with the probe of interest and form a chemical bond therewith.

The bond which forms between E, or a portion thereof, and the probe inmost cases is covelent, or has a covalent character. It is to be noted,however, that the present invention also encompasses other type of bondsor bonding (e.g., hydrogen bonding, ionic bonding, metal coordination,or combinations thereof). One example of the latter is when the E groupcontains a metal complexing agent that can bind a protein through amixed complex: E can be, for instance, a ligand, such as iminodiaceticacid that can bind histidine tagged proteins through Ni mixed complexes.

E can be for example, but is not limited to, isothiocyanates,isocyanates, acylacydes, aldehydes, amines, sulfonylchlorides, epoxides,carbonates, acidfluorides, acidchlorides, acidbromides, acidanhydrides,acylimidazoles, thiols, alkyl halides, maleimides, aziridines andoxiranes.

In another embodiment, E is a phenylboronic acid moiety, which canstrongly complex to biological probes that contains certain polyolmolecules (e.g., 1,2-cis diols or other related compounds). In onepreferred embodiment, E is an electrophilic group that, upon reactionwith a nucleophilic site present in the probe, forms a chemical bondwith the probe. Such activated monomers include, but are not limited to,N-hydroxysuccinimides, tosylates, brosylates, nosylates, mesylates, etc.In other embodiments, the electrophilic group consists of a 3- to5-membered ring which opens upon reaction with the nucleophile. Suchcyclic electrophiles include, but are not limited to, epoxides,oxetanes, aziridines, azetidines, episulfides, 2-oxazolin-5-ones, etc.In still other embodiments, the electrophilic group may be a groupwherein, upon reaction with the nucleophilic probe, an addition reactiontakes place, leading to the formation of a covalent bond between theprobe and the polymer. These electrophilic groups include, but are notlimited to, maleimide derivatives, acetylacetoxy derivatives, etc.

With respect to X, it is to be noted that, when present (i.e., when n isnot equal to zero), X represents some linking group which connects P toE, such as in the case of X linking an unsaturated carbon atom of P toan electrophilic E group. X may be, for example, a substituted orunsubstituted hydrocarbylene or heterohydrocarbylene linker, a heterolinker, etc., including linkers derived from alkyl, amino, aminoalkyl oraminoalkylamido groups. In such instances, m is an integer such as 1, 2,3, 4 or more. In other embodiments (i.e., when n is equal to zero), P isdirectly bound to E.

X is for example chosen from a covalent bond, an optionally substitutedC₁-C₄O alkyl radical optionally interrupted by a (hetero)cycle, thealkyl radical being optionally interrupted by at lest one heteroatom orgroup comprising at least one heteroatom or an optionally substitutedphenyl radical.

In one preferred embodiment, X is a linker generally represented by theformula

wherein n is an integer from about 1 to about 5, and m is an integerfrom about 1 to about 2, 3, 4 or more. In one such embodiment, preferredmonomers include those having an N-hydroxysuccinimide group. Forexample, certain of such monomers may generally be represented by thefollowing formula

wherein

-   R₄ is a hydrogen or an akyl substitutent, and-   R₇ is one or more substituents (i.e., w is 1, 2) selected from the    group consisting of hydrogen substituted or unsubstituted    hydrocarbyl (e.g., alkyl, aryl, heteroalkyl), heterohydrocarbyl,    alkoxy, substituted or unsubstituted aryl, sulphates, thioethers,    ethers, hydroxy, etc.

Generally speaking, R₇ can essentially be any substituent that does notsubstantially decrease the hydrophilic of the water-soluble orwater-dispersible segment in which it is contained. In this regard it isto be noted that a number of substituted succinimide compounds arecommercially available and are suitable for use in the presentinvention.

Among the particularly preferred monomers is includedN-acryloxysuccinimide and 2-(methacryloyloxy)ethylamino N-succinimidvlcarbamate. which are generally represented by compounds of the formula(I)

and formula (II)

wherein

R₄, R₇ and w are as previously defined.

Also preferred are those monomers represented by formulas (III) and (IV)below, wherein the terminal carbonyl-oxo-succinimide group is positionedfurther from the polymer chain backbone by the presence of an aminoalkylor aminoalkylamido linker (i.e., “X”), respectively the compounds offormula (III)

and

(IV)

wherein

R₄, R₇, n and w are as previously defined.

Alternatively, however, monomers such as 2-(methylacryloyloxy)ethylacetoacetate, glycidyl methacrylate (GMA) and4,4-dimethyl-2-vinyl-2-oxazolin-5-one, generally represented by formulas

respectively, may also be employed. R₉ is hydrogen or hydrocarbyl, suchas methyl, ethyl, propyl, etc., as defined herein).

One or more of the above referenced monomers (e.g.,N-acryloxysuccinimide, 2-(methylacryloyloxy)ethyl acetoacetate, glycidylmethacrylate and 4,4-dimethyl-2-vinyl-2-oxazolin-5-one) are commerciallyavailable, for example from Aldrich Chemical Company. Additionally,monomers generally represented by formulas (III) and (IV), above, may beprepared by means common in the art.

It is to be noted that such monomers may advantageously be employed inany of the polymerization processes described herein, includingnitroxide and iniferter initiated systems.

Suitable polymerization monomers and comonomers of the present inventioninclude, but are not limited to, methyl methacrylate, ethyl acrylate,propyl methacrylate (all isomers), butyl methacrylate (all isomers),2-ethylhexyl methacrylate, isobornyl methacrylate, methacrylic acid,benzyl methacrylate, phenyl methacrylate, methacrylonitrile,alpha-methylstyrene, methyl acrylate, ethyl acrylate, propyl acrylate(all isomers), butyl acrylate (all isomers), 2-ethylhexyl acrylate,isobornyl acrylate, acrylic acid, benzyl acrylate, phenyl acrylate,acrylonitrile, styrene, acrylates and styrenes selected from glycidylmethacrylate, 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate(all isomers), hydroxybutyl methacrylate (all isomers),N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate,triethyleneglycol methacrylate, itaconic anhydride, itaconic acid,glycidyl acrylate, 2-hydroxyethyl acrylate, hydroxypropyl acrylate (allisomers), hydroxybutyl acrylate (all isomers), N,N-dimethylaminoethylacrylate, N,N-diethylaminoacrylate, triethyleneglycol acrylate,methacrylamide, N-methylacrylamide, N,N-dimethylacrylamide,N-tert-butylmethacrylamide, N-n-butylmethacrylamide,N-methylolacrylamide, N-ethylolacrylamide, vinyl benzoic acid (allisomers), diethylaminostyrene (all isomers), alpha-methylvinyl benzoicacid (all isomers), diethylamino alpha-methylstyrene (all isomers),p-vinylbenzenesulfonic acid, p-vinylbenzene sulfonic sodium salt,trimethoxysilylpropyl methacrylate, triethoxysilylpropyl methacrylate,tributoxysilylpropyl methacrylate, dimethoxymethylsilylpropylmethacrylate, diethoxymethylsilylpropyl methacrylate,dibutoxymethylsilylpropyl methacrylate, diisopropyoxymethylsilylpropylmethacrylate, dimethoxysilylpropyl methacrylate, diethoxysilylpropylmethacrylate, dibutoxysilylpropyl methacrylate, diisopropoxysilylpropylmethacrylate, trimethoxysilylpropyl acrylate, triethoxysilylpropylacrylate, tributoxysilylpropyl acrylate, dimethoxymethylsilylpropylacrylate, diethoxymethylsilylpropyl acrylate, dibutoxymethylsilylpropylacrylate, diisopropoxymethylsilylpropyl acrylate, dimethoxysilylpropylacrylate, diethoxysilylpropyl acrylate, dibutoxysilylpropyl acrylate,diisopropoxysilylpropyl acrylate, vinyl acetate, vinyl butyrate, vinylbenzoate, vinyl chloride, vinyl flouride, vinyl bromide, maleicanhydride, N-phenyl maleimide, N-butylmaleimide, N-vinylpyrrolidone,N-vinylcarbazole, betaines, sulfobetaines, carboxybetaines,phosphobetaines, butadiene, isoprene, chloroprene, ethylene, propylene,1,5-hexadienes, 1,4-hexadienes, 1,3-butadienes, and 1,4-pentadienes.

Additional suitable polymerizable monomers and comonomers include, butare not limited to, vinyl acetate, vinyl alcohol, vinylamine,N-alkylvinylamine, allylamine, N-alkylallylamine, diallylamine,N-alkyldiallylamine, alkylenimine, acrylic acids, alkylacrylates,acrylamides, methacrlic acids, maleic anhydride, alkylmethacrylates,n-vinyl formamide, vinyl ethers, vinyl naphthalene, vinyl pyridine,vinyl sulfonates, ethylvinylbenzene, aminostyrene, vinylbiphenyl,vinylanisole, vinylimidazolyl, vinylpyridinyl,dimethylaminomethystyrene, trimethylammonium ethyl methacrylate,trimethylammonium ethyl acrylate, dimethylamino propylacrylamide,trimethylammonium ethylacrylate, trimethylammonium ethyl methacrylate,trimethylammonium propyl acrylamide, dodecyl acrylate, octadecylacrylate, and octadecyl methacrylate.

“Betaine”, as used herein, refers to a general class of salt compounds,especially zwitterionic compounds, and include polybetaines.Representative examples of betaines which can be used with the presentinvention include:N,N-dimethyl-N-acryloyloxyethyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(3-sulfopropyl)-ammonium betaine,N,N-dimethyl-N-acrylamidopropyl-N-(2-carboxymethyl)-ammonium betaine,2-(methylthio)ethyl methacryloyl-S-(sulfopropyl)-sulfonium betaine,2-[(2-acryloylethyl)dimethylammonio]ethyl 2-methyl phosphate,2-(acryloyloxyethyl)-2′-(trimethylammonium)ethyl phosphate,[(2-acryloylethyl)-dimethylammonio]methyl phosphonic acid,2-methacryloyloxyethyl phosphorylcholine (MPC),2-[(3-acrylamidopropyl)dimethylammonio]ethyl 2′-isopropyl phosphate(AAPI), 1-vinyl-3-(3-sulfopropyl)imidazolium hydroxide,(2-acryloxyethyl)carboxymethyl methylsulfonium chloride,1-(3-sulfopropyl)-2-vinylpyridinium betaine,N-(4-sulfobutyl)-N-methyl-N,N-diallylamine ammonium betaine (MDABS),N,N-diallyl-N-methyl-N-(2-sulfoethyl)ammonium betaine, and the like.

It is to be understood, that the above described functional monomers,especially monomers containing basic amino groups, can also be used inform of their corresponding salts. For example acrylates, methacrylatesor styrenes containing amino groups can be used as salts with organic orinorganic acids or by way of quaternisation with known alkylation agentslike benzyl chloride. The salt formation can also be done as asubsequent reaction on the preformed block copolymer with appropriatereagents. In another embodiment, the salt formation is carried out insitu in compositions or formulations, for example by reacting a blockcopolymer with basic or acidic groups with appropriate neutralisationagents during the preparation of a pigment concentrate.

The grafted polymers formed on the surface of the halogenated polymersubstrate form thin layers of 5 nm to 100 μm, preferably 10 nm to 200 nmand distinguish by a low polydisperisty which is <3.

The layer thickness of the polymers formed on the surface is dependenton the parameters like solvents, concentration of reactands, temperatureand/or reaction time.

If necessary, these polymers may be present in form of polymer brushes,i.e. in form of chains which are oriented perpendicular to the surface.

“Polymer brushes,” as the name suggests, contain polymer chains, one endof which is directly or indirectly tethered to a surface and another endof which is free to extend from the surface, somewhat analogous to thebristles of a brush.

Covalent attachment of polymers to form polymer brushes is commonlyachieved by “grafting to” and “grafting from” techniques. “Grafting to”techniques involve tethering pre-formed end-functionalized polymerchains to a suitable substrate under appropriate conditions. “Graftingfrom” techniques, on the other hand, involve covalently immobilizinginitiators on the substrate surface, followed by surface initiatedpolymerization to generate the polymer brushes.

Each of these techniques involves the attachment of a species (e.g., apolymer or an initiator) to a surface, which may be carried out using anumber of techniques that are known in the art.

As noted above, in the “grafting from” process once an initiator isattached to the surface, a polymerization reaction is then conducted tocreate a surface bound polymer. Various polymerization reactions may beemployed, including various condensations, anionic, cationic and radicalpolymerization methods. These and other methods may be used topolymerize a host of monomers and monomer combinations.

Specific examples of radical polymerization processes arecontrolled/“living” radical polymerizations such as metal-catalyzed atomtransfer radical polymerization (ATRP), stable free-radicalpolymerization (SFRP), nitroxide-mediated processes (NMP), anddegenerative transfer (e.g., reversible addition-fragmentation chaintransfer (RAFT)) processes, among others. The advantages of using a“living” free radical system for polymer brush creation include controlover the brush thickness via control of molecular weight and narrowpolydispersities, and the ability to prepare block copolymers by thesequential activation of a dormant chain end in the presence ofdifferent monomers. These methods are well-detailed in the literatureand are described, for example, in an article by Pyun and Matyjaszewski,“Synthesis of Nanocomposite Organic/Inorganic Hybrid Materials UsingControlled/”Living “Radical Polymerization,” Chem. Mater., 2001, 13,3436-3448, the contents of which are incorporated by reference in itsentirety.

If necessary, the first polymerization may be interrupted and a furtherpolymerisation may be started with a new monomer in order to form blockpolymers.

The term polymer comprises oligomers, cooligomers, polymers orcopolymers, such as block, multi-block, star, gradient, random, comb,hyperbranched and dendritic copolymers as well as graft copolymers. Theblock copolymer unit A contains at least two repeating units (x≧2) ofpolymerizable aliphatic monomers having one or more olefinic doublebonds. The block copolymer unit B contains at least one polymerizablealiphatic monomer unit (y≧0) having one or more olefinic double bonds.

The modified halogenated polymer substrate prepared according to theprocess of the present invention represents a further embodiment of thepresent invention.

The modified halogenated polymer can be represented by the followingformula:

-   (1) HalPol-[In-A_(x)-B_(y)-C_(z)-Z]_(n), wherein-   A, B, C represent monomer-oligomer or polymer fragments, which can    be arranged in block or statstically;-   Z is halogen which is positioned at the end of each polymer brush as    end group derived from ATRP;

represents the halogenated polymer substrate;

-   In represents the fragment of a polymerisation initiator capable of    initiating polymerisation of ethylenically unsaturated monomers in    the presence of a catalyst which activates controlled radical    polymerisation;-   x represents a numeral greater than one and defines the number of    repeating units in A;-   y represents zero or a numeral greater than zero and defines the    number of monomer, oligopolymer or polymer repeating units in B;-   z represents zero or a numeral greater than zero and defines the    number of monomer, oligopolymer or polymer repeating units in C;-   n is one or a numeral greater than one which defines the number of    groups of the partial formula (1a) In-(A_(x)-B_(y)-C_(z)-Z)—.

The subunits A, B, and C can be further subdivided into the generalformula (1 b) P-[X]_(n)-E, wherein

P, X, E and n are defined as above.

In the context of the description of the present invention, the termalkyl comprises methyl, ethyl and the isomers of propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl. An example ofaryl-substituted alkyl is benzyl. Examples of alkoxy are methoxy, ethoxyand the isomers of propoxy and butoxy. Examples of alkenyl are vinyl andallyl. An example of alkylene is ethylene, n-propylene, 1,2- or1,3-propylene.

Some examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, methylcyclopentyl, dimethylcyclopentyl and methylcyclohexyl.Examples of substituted cycloalkyl are methyl-, dimethyl-, trimethyl-,methoxy-, dimethoxy-, trimethoxy-, trifluoromethyl-,bis-trifluoromethyl- and tris-trifluoromethyl-substituted cyclopentyland cyclohexyl.

Examples of aryl are phenyl and naphthyl. Examples of aryloxy arephenoxy and naphthyloxy. Examples of substituted aryl are methyl-,dimethyl-, trimethyl-, methoxy-, dimethoxy-, trimethoxy-,trifluoromethyl-, bis-trifluoromethyl- ortris-trifluoromethyl-substituted phenyl. An example of aralkyl isbenzyl. Examples of substituted aralkyl are methyl-, dimethyl-,trimethyl-, methoxy-, dimethoxy-, trimethoxy-, trifluoromethyl-,bis-trifluoromethyl or tris-trifluoromethyl-substituted benzyl.

Some examples of an aliphatic carboxylic acid are acetic, propionic orbutyric acid. An example of a cycloaliphatic carboxylic acid iscyclohexanoic acid. An example of an aromatic carboxylic acid is benzoicacid. An example of a phosphorus-containing acid is methylphosphonicacid. An example of an aliphatic dicarboxylic acid is malonyl, maleoylor succinyl. An example of an aromatic dicarboxylic acid is phthaloyl.

The term heterocycloalkyl embraces within the given structure one or twoand heterocyclic groups having one to four heteroatoms selected from thegroup consisting of nitrogen, sulphur and oxygen. Some examples ofheterocycloalkyl are tetrahydrofuryl, pyrrolidinyl, piperazinyl andtetrahydrothienyl. Some examples of heteroaryl are furyl, thienyl,pyrrolyl, pyridyl and pyrimidinyl.

An example of a monovalent silyl radical is trimethylsilyl.

The modified halogenated polymer substrate according to the presentinvention can be used for many applications.

Sensing Devices:

The first requirement for an analytical or sensing device, which allowsspecific detection or recognition, is the resistance of the devicesurface towards non-specific adsorption. This requirement can befulfilled by the copolymers described above. The second requirement isthe introduction of functional groups, hereafter called recognitionunits, that allow specific interaction with selected components of theanalyte. Examples are: Recognition units that induce physico-chemicaladsorption of a molecule for the subsequent analytical or sensingdetection. Examples of the recognition units are any structural unitable to recognize and which will specifically bind (complex) moleculesto be analyzed during the sensing step (called target molecules) such asfor example organic molecules, biomarkers, metabolites, peptides,proteins, oligonucleotides, DNA or RNA fragments, carbohydrates orfragments thereof. The interaction of the recognition unit and thetarget molecule will be accomplished by hydrogen bonding, electrostaticinteractions, van der Waals forces, C═C interactions, hydrophopicinteractions, metal coordination, or combinations thereof.

Examples of recognition units comprise esters, amides, urethanes,carbamates, imides like maleimide or succinimidyl, vinylsulfones,conjugated C═C double bonds, epoxides, aldehydes, ketones, alcohols,ethers, amines, nitrogroups, sulfoxides, sulfones, sulfonamides, thiols,disulfides, silane or siloxane functionalities. These recognition unitscan react with functional groups of the target molecules.

Recognition units that are able to bind to receptors on the surfaces ofcells: a target molecule may be bound to the recognition unit directlyby reaction. An example is the reaction of a cysteine-containing peptideto a vinylsulfone recognition unit. The case of the peptide recognitionunit binding to receptors on the surface of a cell can be particularlyinteresting, e.g. in analysis of cellular behavior or in the therapeuticmanipulation of cell behavior in a culture system or upon an implant.

Recognition units that are able to bind specifically to a bioactivetarget moiety: examples of such targets include antigens, proteins,enzymes, oligonucleotides, DNA and RNA fragments, carbohydrates as forexample glucose and other groups or molecules provided they are able tointeract specifically with the recognition unit in the subsequentanalytical or sensing assay.

Recognition units that are able to form stable complexes with a cation.In a second step the cation will form a complex either with the targetmolecule directly through a suitable functionality. Examples for therecognition unit include carboxylate, amide, phosphate, phosphonate,nitrilo triacetic acid and other known groups that are able to chelatecations. Examples for the cations include Mg(II), Ti(IV), Co(III),Co(VI), Cu(II), Zn(II), Zr(IV), Hf(IV), V(V), Nb(V), Ta(V), Cr(III),Cr(VI), Mo(VI) and other cations known to form stable complexes withchelating ligands.

Many interesting recognition units in the bioanalysis of cellularresponses are peptides. In such cases, the peptides may be coupled tothe modified halogenated polymer surface (1). The peptide may be boundto the modified halogenated polymer surface (1) through a number ofmeans, including reaction to a cysteine residue incorporated within thepeptide. Cysteine residues are rarely involved in cell adhesiondirectly. As such, few cell adhesion peptides comprise a cysteineresidue, and thus a cysteine residue that is incorporated for thepurpose of coupling of the peptide will be the unique cysteine residuefor coupling. While other approaches are possible, the preferred methodis coupling of the peptide to the multifunctional polymer through acysteine residue on the polymer. Other bioactive features can also beincorporated, e.g. adhesion proteins, growth factor proteins, cytokineproteins, chemokine proteins, and the like. Functionalized surfaces canbe used in bioanalytical systems involving cells, in which some effecterof cell function is the measured feature. A test fluid may contain ananalyte, to which the response of cells is sought. The cellular responsemay be used in as a measure of the presence or the activity of theanalyte. Alternatively, the cellular response per se may be theknowledge that is sought, e.g. the migration response of a particularcell type to a growth factor, when the cells are migrating upon aparticular adhesive substrate. The collection of such scientificinformation is of significant value in the screening of the activity ofdrug candidates, particularly when higher order cellular responses suchas adhesion, migration, and cell-cell interactions are targeted.

Functionalized surfaces can be used in therapeutic systems involvingcells, in which cells are cultured for later therapeutic use. In currenttherapeutic systems, cultured cells are sometimes used. Examples are inthe culture of chondrocytes for transplantation in articular cartilagedefects in the knee or in the culture of endothelial cells fortransplantation in vascular grafts. In such cases, modulation andmanipulation of the phenotype of the cells is of prime interest.

Functionalized surfaces can be used in medical devices. In general amedical device is any article, natural or synthetic, that comprises allor part of a living structure which performs, augments, protects orreplaces a natural function and that is substantially compatible withthe body.

Any shaped article can be made using the compositions of the invention.For example, articles suitable for contact with bodily fluids, such asmedical devices can be made using the compositions described herein. Theduration of contact may be short, for example, as with surgicalinstruments or long term use articles such as implants. The medicaldevices include, without limitation, catheters, guide wires, vascularstents, micro-particles, electronic leads, probes, sensors, drug depots,transdermal patches, vascular patches, blood bags, and tubing. Themedical device can be an implanted device, percutaneous device, orcutaneous device. Implanted devices include articles that are fullyimplanted in a patient, i.e., are completely internal. Percutaneousdevices include items that penetrate the skin, thereby extending fromoutside the body into the body. Cutaneous devices are usedsuperficially. Implanted devices include, without limitation, prosthesessuch as pacemakers, electrical leads such as pacing leads,defibrillarors, artificial hearts, ventricular assist devices,anatomical reconstruction prostheses such as breast implants, artificialheart valves, heart valve stents, pericardial patches, surgical patches,coronary stents, vascular grafts, vascular and structural stents,vascular or cardiovascular shunts, biological conduits, pledges,sutures, annuloplasty rings, stents, staples, valved grafts, dermalgrafts for wound healing, orthopedic spinal implants, orthopedic pins,intrauterine devices, urinary stents, maxial facial reconstructionplating, dental implants, intraocular lenses, clips, sternal wires,bone, skin, ligaments, tendons, and combination thereof. Percutaneousdevices include, without limitation, catheters or various types,cannulas, drainage tubes such as chest tubes, surgical instruments suchas forceps, retractors, needles, and gloves, and catheter cuffs.Cutaneous devices include, without limitation, burn dressings, wounddressings and dental hardware, such as bridge supports and bracingcomponents.

Functionalzed surfaces can be used in therapeutic systems involvingcells, in which the cells are cultured and used in contact with thesurface. As an example of this situation, bioreactors are used in someextracorporal therapeutic systems, such as cultured hepatocytes used todetoxify blood in acute hepatic failure patients. In such cases, onewants to maintain the hepatocytes in the reactor in a functional,differentiated state. The adhesive interactions between the cells andtheir substrate are thought to play an important role in theseinteractions, and thus the technology of this invention provides a meansby which to control these responses.

Functionalized surfaces can be used in therapeutic systems involvingcells, in which the functionalized surfaces are a component of animplant. The interactions between cells in an implant environment andthe surface of an implant may play a controlling role in determining thebiocompatibility of an implant. For example, on the surface of a stentimplanted within the coronary artery, the presence of blood platelets isnot desirable and may lead to in-stent restenosis. As such, it would bedesirable to prevent the attachment of blood platelets to the stentsurface.

The materials described here have a variety of applications in the areaof substrates or devices (called ‘chips’ in the general sense) foranalytical or sensing purposes. In particular, they are suited for thesurface treatment of chips intended to be used in analytical or sensingapplications where the aim is specific detection of biologically ormedically relevant molecules such as peptides, proteins,oligonucleotides, DNA or RNA fragments or generally any type ofantigen-antibody or key-loch type of assays. Particularly if the analytecontains a variety of molecules or ionic species, and if the aim iseither to specifically detect one molecule or ion out of the manycomponents or several molecules or ions out of the many components, theinvention provides a suitable basis for producing the necessaryproperties of the chip surface: 1) the ability to withstand non-specificadsorption and 2) the ability to introduce in a controlled way a certainconcentration of recognition entities, which will during the analyticalor sensing operation interact specifically with the target molecules orions in the analyte. If combined with suitable analytical or sensordetection methods, the invention provides the feasibility to producechips that have both high specificity and high detection sensitivity inany type of analytical or sensing assay, in particular in bioaffinitytype of assays.

The materials described here additionally have a variety of applicationsin the area of substrates or devices which are not “chip” basedapplications. In particular, for use in analytical or sensingapplications where the aim is specific detection of biologically ormedically relevent molecules such as peptides, proteins,oligonucleotides, DNA or RNA fragments or generally any type ofantigen-antibody or key-loch type of assays.

The methods can be applied to chips for any type of qualitative,semiquantitative or quantitative analytical or sensing assay.Particularly suitable detection techniques to be combined with chipsinclude:

-   1) The optical waveguide technique, where the evanescent field is    used to interact with and detect the amount of target molecules    adsorbed to the chips surface. The technique relies on incoupling    white or monochromatic light into a waveguiding layer through an    optical coupling element, preferably a diffraction grating or    holographic structure.-   2) Fluorescence spectroscopy or microscopy where fluorescently    labeled target molecules are quantitatively analyzed by measuring    the intensity of the fluorescence light.-   3) Combination of 1) and 2), where the evanescent optical field is    used to excite the fluorescence tags of target or tracer molecules    adsorbed onto the chip surface modified. The fluorescence is    detected using a fluorescence detector situated on the side opposite    to the liquid flow cell.-   4) The Surface Plasmon Resonance Technique (SPR) where the    interaction of surface plasmons in thin metal films resonance    condition, i.e., the resonant incidence angle for the escitation of    a surface plasmon in a thin metal film, is changed upon molecular    adsorption or desorption into/from the metal film, due to the    resulting change of the effective refractive index.-   5) Ultraviolet or Visible (UV/VIS) Spectroscopy where the adsorption    at a particular characteristic wavelength is used to quantitfy the    amount of target molecules adsorbed or attached to the modified    surface.-   6) Infrared Techniques such as Fourier Transform Infrared (FTIR)    Spectroscopy, where the excitation of atomic or molecular vibrations    in the infrared region is used to detect and quantify target    molecules that have previously been adsorbed or attached to the    surface modified chips. Surface or interface sensitive forms of IR    spectroscopy such as Attenuated Total Reflection Spectroscopy    (ATR-FTIR) or Infrared Reflection-Adsorption Spectroscopy (IRAS) are    particularly suitable techniques.-   7) Raman Spectroscopy (RS) to detect specific vibrational levels in    the molecule adsorbed or attached onto the modified chip surface.    Surface- or interface-sensitive types of RS are particularly    suitable, e.g. Surface Enhanced Raman Spectroscopy (SERS).-   8) Electrochemical techniques where for example the current or    charge for the reduction or oxidation of a particular target    molecule or part of that molecule is measured at a given potential.    Chip based devices can also be assayed with standard fluorescence or    adsorption techniques in which excitation is through light reflected    off the substrate surface as opposed to the evanescent field    interaction.

Other analytical or bioanalytical device surfaces can be used forqualitative, semiquantitative or quantitative analytical or sensingassays. Non “chip” based substrates also includes fiberoptic substrates.In the case of fiberoptics, techniques as described for “chip”substrates are applicable. For other non “chip” based substrates whichdo not support evanescent field excitation or are not a “chip”, suitabletechniques are described below.

-   1) Fluorescence spectroscopy or microscopy where fluorescently    labeled target molecules are quantitatively analyzed by measuring    the intensity of the fluorescence light. The fluorescence is    detected using standard detectors positioned either for    transmission, or more preferably, for reflection based detection    methods.-   2) Adsorption spectroscopy where the adsorption at a particular    characteristic wavelength is used to quantitfy the amount of target    molecules adsorbed or attached to the surface modified according to    the invention through reflection or transmission techniques. For    simple assay formats such as lateral flow assays, the detection by    visual inspection of a color change in the assay region.-   3) Infrared Techniques such as Fourier Transform Infrared (FTIR)    Spectroscopy, where the excitation of atomic or molecular vibrations    in the infrared region is used to detect and quantify target    molecules that have previously been adsorbed or attached to the    modified chip surface. Surface or interface sensitive forms of IR    spectroscopy such as Infrared Reflection-Adsorption Spectroscopy    (IRAS) are particularly suitable techniques.-   4) Electrochemical techniques where for example the current or    charge for the reduction or oxidation of a particular target    molecule or part of that molecule is measured at a given potential.

The analytical or sensor chips can be used in a variety of ways.

Non-modified and modified copolymers can be adsorbed onto suitablesurfaces either in pure form or as mixtures. The optimum choice dependson the type and concentration of the target molecules and on the type ofdetection technique. Furthermore, the technique is particularly suitedfor the modification of chips to be used in assays where multipleanalytes are determined on one chip, either sequentially orsimultaneously.

Examples are microarrays for multipurpose DNA and RNA bioaffinityanalysis ‘Genomics Chips’, for protein recognition and analysis based onsets of antibody-antigen recognition and analyze (Proteomics Chips).Such techniques are particularly efficient for the analysis of amultitude of components on one miniaturized chip for applications inbiomedical, diagnostic DNA/RNA, or protein sensors or for the purpose ofestablishing extended libraries in genomics and proteomics.

From the viewpoint of the detection step, there are two basicalternatives:

-   1) In a type of batch process where the chip is functionalized. In a    fluid manifold, one or several analytes and reagents are locally    applied to the chip surface. After awaiting the completion or near    completion of the bioaffinity reaction (incubation step), the chip    is washed in a buffer and analyzed using one or a combination of the    methods described above.-   2) In a continuous process where the chip is functionalized and is    part of a gaseous or liquid cell or flow-through cell. The    conditioning of the surface can be done in a continuous and    continuously monitored process within that liquid or flow-through    cell, followed by in situ monitoring of the signal due to the    specific interaction and adsorption or attachment of the specific    target molecule in the analyte solution. The original surface of the    chip may afterwards be restored/regenerated again and conditioned    for the immediately following next bioaffinity assay. This may be    repeated many times.

In a related but different area, the surface treatment of chips hasapplications in biosensors, where the aim is to attach and organizeliving cells in a defined manner on such chips. Since protein adsorptionand cell attachment is closely related, this opens the possibility toorganize cells on chips in defined way.

The detection of specific areas of the pattern can be localized to thespecific areas, or can be performed for multiple specific areassimultaneously. In general, an important aspect is the sequential orsimultaneous determination of multiple analytes in one or more liquidsamples, where the patterned surface is used in microarray assays forthe determination of analytes of the group formed of peptides, proteins,antibodies or antigens, receptors or their ligands, chelators or“histidin tag components”, oligonucleotides, polynucleotides, DNA, andRNA fragments, enzymes, enzyme cofactors or inhibitors, lectins,carbohydrates.

In summary, the materials and methods described herein can be used inmany application areas, e.g., for the quantitative or qualitativedetermination of chemical, biochemical or biological analytes inscreening assays in pharmacological research, combinatorial chemistry,clinical or preclinical development, for real-time binding studies orthe determination of kinetic parameters in affinity screening or inresearch, for DNA and RNA analytics and the determination of genomic orproteomic differences in the genome, such as single nucleotidepolymorphisms, for the determination of protein-DNA interactions, forthe determination of regulation mechanisms for mRNA expression andprotein (bio)synthesis, for toxicological studies and the determinationof expression profiles, especially for the determination of biologicalor chemical markers, such as mRNA, proteins, peptides or low molecularorganic (messenger) compounds, for the determination of antigens,pathogens or bacteria in pharmacological product research anddevelopment, human and veterinary diagnostics, agrochemical productresearch and development, symptomatic and presymptomatic plantdiagnostics, for patient stratification in pharmaceutical productdevelopment and for the therapeutic drug selection, for thedetermination of pathogens, harmful compounds or germs, especially ofsalmonella, prions, viruses and bacteria, especially in nutritional andenvironmental analytics.

There is a need to improve the selectivity and sensitivity ofbioaffinity and diagnostic sensors, especially for use in screeningassays and libraries for DNA/RNA and proteins. A common approach todiagnostic sensor design involves the measurement of the specificbinding of a particular component of a physiological sample. Typically,physiological samples of interest (e.g. blood samples) are complexmixtures of many components that all interact to varying degrees withsurfaces of diagnostic sensors. However, the aim of a diagnostic sensoris to probe only the specific interaction of one component whileminimizing all other unrelated interactions. In the case of sensors incontact with blood, proteins, glycoproteins and/or saccharides, as wellas cells, often adsorb non-specifically onto the sensor surface. Thisimpairs both selectivity and sensitivity, two highly importantperformance criteria in bioaffinity sensors.

As outlined above, it is possible to use reactive monomers whichdirectly yield a polyfunctional polymer monolayer according to theinvention. Alternatively, monomers can be chosen which carry a precursorof the functional group to be used on the final surface, e.g. an acidchloride or an acid anhydride. They can subsequently be transformed toreactive groups, e.g. NHS ester or glycidylester groups, which allow aninteraction of the polymer with sample or probe molecules under thedesired conditions.

Thus, all polymerizable monomers are suitable for the purposes of thepresent invention, as long as they can be combined with, or comprise,functional groups necessary to allow an interaction of the polymer withthe sample molecules or probe molecules.

Functional groups which can be used for the purposes of the presentinvention are preferably chosen according to the molecules with which aninteraction is to be achieved. The interaction can be directed to onesingle type of sample molecule, or to a variety of sample molecules.Since one important application of the present invention is thedetection of specific molecules in biological samples, the functionalgroups present within the polymer brushes will preferably interact withnatural or synthetic biomolecules which are capable of specificallyinteracting with the molecules in biological samples, leading to theirdetection. Suitable functional moieties will preferably be able to reactwith nucleic acids and derivatives thereof; such as DNA, RNA or PNA,e.g. oligonucleotides or aptamers, saccharides and polysaccharides,proteins including glycosidically modified proteins or antibodies,enzymes, cytokines, chemokines, peptidhormones or antibiotics orpeptides or labeled derivatives thereof.

Since most of the probe molecules, especially in biological or medicalapplications, comprise sterically unhindered nucleophilic moieties,preferred interactions with the polymer brushes comprise nucleophilicsubstitution or addition reactions leading to a covalent bond betweenthe polymer chains and the sample or probe molecules.

With appropriate functional groups present in the polymer brushes, thepolymer monolayers of the present invention can also be used inseparation methods, e.g. as a stationary phase in chromatographicapplications.

Preferred functional groups can be chosen from prior art literature withrespect to the classes of molecules which are to be immobilized andaccording to the other requirements (reaction time, temperature, pHvalue) as described above. Examples for suitable groups are so-calledactive or reactive esters as N-hydroxy succinimides (NHS-esters),epoxides, preferably glycidyl derivatives, isothiocyanates, isocyanates,azides, carboxylic acid groups or maleinimides.

As preferred functional monomers which directly result in apolyfunctional polymer monolayer, the following compounds can beemployed for the purposes of the present invention: acrylic ormethacrylic acid N-hydroxysuccinimides, N-methacryloyl-6-aminopropanoicacid hydroxysuccinimide ester, N-methacryloyl-6-aminocapronic acidhydroxysuccinimide ester or acrylic or methacryl acid glycidyl esters.

Depending on the application, there is the possibility of providing apolymer brush with a combination of two or more different functionalgroups, e.g. by carrying out the polymerization leading to the polymerchains in the presence of different types of functionalized monomers.Alternatively, the functional groups may be identical.

For the detection of a successful immobilization of sample or probemolecules on a polymer monolayer, a variety of techniques can beapplied. In particular, it has been found that the polymer layers of thepresent invention undergo a significant increase in their thicknesswhich can be detected with suitable methods, e.g. ellipsometry. Masssensitive methods may also be applied.

If nucleic acids, for example oligonucleotides with a desired nucleotidesequence or DNA molecules in a biological sample are to be analyzed,synthetic oligonucleotide single strands can be reacted with the polymermonolayer.

Before the thus prepared surface is used in a hybridization reaction,unreacted functional groups are deactivated via addition of suitablenucleophiles, preferably C.sub.1 C.sub.4 amines, such as simple primaryalkylamines (e.g. propyl or butyl amine), secondary amines(diethylamine) or amino acids (glycin).

Upon exposure to a mixture of oligonucleotide single strands, e.g. asobtained from PCR, which are labeled, only those surface areas whichprovide synthetic strands as probes complementary to the PCR productwill show a detectable signal upon scanning due to hybridization. Inorder to facilitate the parallel detection of different oligonucleotidesequences, printing techniques can be used which allow the separation ofthe sensor surface into areas where different types of syntheticoligonucleotide probes are presented to the test solution.

The term “hybridization” as used in accordance with the presentinvention may relate to stringent or non-stringent conditions.

The nucleic acids to be analyzed may originate from a DNA library or agenomic library, including synthetic and semisynthetic nucleic acidlibraries. Preferably, the nucleic acid library comprisesoligonucleotides.

In order to facilitate their detection in an immobilized state, thenucleic acid molecules should preferably be labeled. Suitable labelsinclude radioactive, fluorescent, phosphorescent, bioluminescent orchemoluminescent labels, an enzyme, an antibody or a functional fragmentor functional derivative thereof, biotin, avidin or streptavidin.

Antibodies may include, but are not limited to, polyclonal, monoclonal,chimeric or single chain antibodies or functional fragments orderivatives of such antibodies.

Depending on the labeling method applied, the detection can be effectedby methods known in the art, e.g. via laser scanning or use of CCDcameras.

Also comprised by the present invention are methods where detection isindirectly effected.

A further application of the polymer monolayers according to theinvention lies in the field of affinity chromatography, e.g. for thepurification of substances. For this purpose, polymer brushes withidentical functional groups or probe molecules are preferably used,which are contacted with a sample. After the desired substance has beenimmobilized by the polymer brush, unbound material can be removed, e.g.in a washing step. With suitable eluents, the purified substance canthen be separated from the affinity matrix.

Preferred substances which may be immobilized on such a matrix arenucleic acid molecules, peptides or polypeptides (proteins, enzymes) (orcomplexes thereof, such as antibodies, functional fragments orderivatives thereof), saccharides or polysaccharides.

A regeneration of the surfaces after the immobilization has taken placeis possible, but single uses are preferred in order to ensure thequality of results.

With the present invention, different types of samples can be analyzedwith an increased precision and/or reduced need of space in serial aswell as parallel detection methods. The sensor surfaces according to theinvention can therefore serve in diagnostical instruments or othermedical applications, e.g. for the detection of components inphysiological fluids, such as blood, serum, sputum etc.

Sensors

The sensors of the present invention (i.e., the polymer brush with aprobe attached) can also be utilized in a multi-step or “sandwich” assayformat, wherein a number of biomolecule targets can be applied oranalyzed in sequential fashion. This approach may be useful toimmobilize a protein probe for the desired biomolecule target. It mayalso be applied as a form of signal enhancement if the secondary,tertiary, etc. biomolecules serve to increase the number of signalreporter molecules (i.e., fluorophores).

The sensors can be used to analyze biological samples such as blood,plasma, urine, saliva, tears, mucuous derivatives, semen, stool samples,tissue samples, tissue swabs and combinations thereof.

Sensors in which the tethered probes are polypeptides can be used, forexample, to screen or characterize populations of antibodies havingspecific binding affinity for a particular target antigen or todetermine if a ligand had affinity for a particular receptor, accordingto procedures described generally in Leuking et al., Anal. Biochem.,1991, 270(1):103 111. Target polypeptides can be labeled, e.g.,fluorescently or with an enzyme such as alkaline phosphatase, or radiolabeling for easy detection.

Probes

A wide variety of biological probes can be employed in connection withthe present invention. In general, the probe molecule is preferablysubstantially selective for one or more biological molecules ofinterest. The degree of selectivity will vary depending on theparticular application at hand, and can generally be selected and/oroptimized by a person of skill in the art.

The probe molecules can be bonded to the functional group-bearingpolymer segments using conventional coupling techniques (an example ofwhich is further described herein below under the heading“Application”). The probes may be attached using covalently ornoncovalently (e.g., physical binding such as electrostatic,hydrophobic, affinity binding, or hydrogen bonding, among others).

Typical polymer brushes functionalities that are useful to covalentlyattach probes are chosen among hydroxyl, carboxyl, aldehyde, amino,isocyanate, isothiocyanate, azlactone, acetylacetonate, epoxy, oxirane,carbonate sulfonyl ester (such as mesityl or tolyl esters), acyl azide,activated esters (such as N(hydroxy)succinimide esters), O-acyliso-ureaintermediates from COOH-carbodiimide adducts, fluoro-aryle, imidoester,anhydride, haloacetyl, alkyliodide, thiol, disulfide, maleimide,aziridine, acryloyl, diazo-alkane, diazo-acetyl, di-azonium, and thelike. These may be provided by copolymerizing functional monomers suchas 2-hydroethyl(meth)acrylate, hydroxyethyl(meth)acrylamide,hydroxyethyl-N(methyl)(meth)acrylamide, (meth)acrylic acid,2-aminoethyl(meth)acrylate, amino-protected monomers such as maleimidoderivatives of amino-functional monomers, 3-isopropenyl,α,α-dimethylbenzylisocyanate, 2-isocyanato-ethylmethacrylate,4,4-dimethyl-2-vinyl-2-oxazoline-5-one,acetylacetonate-ethylmethacrylate, and glycidylmethacrylate.

Post derivatization of polymer brushes proves also to be efficient.Typical methods include activation of —OH functionalized groups with,for example phosgene, thiophosgene, 4-methyl-phenyl sulfonylchoride,methylsulfonylchloride, and carbonyl di-imidazole. Activation ofcarboxylic groups can be performed using carbodiimides, such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, or1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, among others. Aldehydegroups can be synthesized from the periodate-mediated oxidation ofvicinal —OH, obtained from hydrolysis of epoxy functional brushes.Alternatively, aldehyde groups are attached by reaction of bis-aldehydes(e.g, glutaraldehyde) onto aminomodified polymer brushes.Amino-functional brushes can also be prepared by reacting diaminocompound on aminoreactive brushes, such as N(hydroxy)succinimide estersof carboxylates brushes. (Other state-of-the-art coupling chemistries,such as described in Bioconjuguate Techniques, Greg. T. Hermanson,Academic Press, 1996, are also applicable and are incorporated herein byreference.)

Examples of probes used herein include: acetylcholin receptor proteins,histocompatibility antigens, ribonucleic acids, basement membraneproteins, immunoglobulin classes and subclasses, myeloma proteinreceptors, complement components, myelin proteins, and various hormones,vitamines and their receptor components as well as geneticallyengineered proteins, nucleic acids and derivatives of, such as DNA, RNAor peptide nucleic acids, oligonucleotides or aptamers, polysaccharides,proteins including glycosidically modified proteins or antibodies,enzymes, cytokines, chemokines, peptidhormones or antibioticsor peptidesor labeled derivatives thereof. The probe may be selected from the groupconsisting of natural or synthetic extracellular proteins, antibodies,antibody fragments, cell adhesion molecules, fragments of cell adhesionmolecules, growth factors, cytokines, peptides, sugars, carbohydrates,polysaccharides, lipids, sterols, fatty acids and combinations thereof.More particularly, biomolecules that are contemplated as being suitablefor linking with the functionalized monomers or polymer segmentscontemplated herein in accordance with the invention include, forexample:

Bioadhesives, including fibrin; fibroin; Mytilus edulis foot protein(mefpi , “mussel adhesive protein”); other mussel's adhesive proteins;proteins and peptides with glycine-rich blocks; proteins and peptideswith poly-alanine blocks; and silks.

Cell Attachment Factors (biomolecules that mediate attachment andspreading of cells onto biological surfaces or other cells and tissues)including molecules participating in cell-matrix and cell-cellinteraction during vertebrate development, neogenesis, regeneration andrepair, such as molecules on the outer surface of cells like the CDclass of receptors on white blood cells, immunoglobulins andhaemagglutinating proteins, and extracellular matrix molecules/ligandsthat adhere to such cellular molecules, ankyrins; cadherins (Calciumdependent adhesion molecules); connexins; dermatan sulfate; entactin;fibrin; fibronectin; glycolipids; glycophorin; glycoproteins; heparansulfate; heparin sulfate; hyaluronic acid; immunoglobulins; keratansulfate; integrins; laminins; N-CAMs (Calcium independent AdhesiveMolecules); proteoglycans; spektrin; vinculin; and vitronectin.

Biopolymers, including parts of the extracellular matrix whichparticipate in providing tissue resilience, strength, rigidity,integrity, such as alginates; amelogenins; cellulose; chitosan;collagen; gelatins; oligosaccharides; and pectin.

Blood proteins (dissolved or aggregated proteins which normally arepresent whole blood, which participate in a wide range of biologicalprocesses like inflammation, homing of cells, clotting, cell signaling,defence, immune reactions, and metabolism) such as albumin; albumen;cytokines; factor IX; factor V; factor VII; factor VIII; factor X;factor XI; factor XII; factor XIII; hemoglobins (with or without iron);immunoglobulins (antibodies); fibrin; platelet derived growth factors(PDGFs); plasminogen; thrombospondin; and transferrin.

Enzymes (any protein or peptide that has a specific catalytic effect onone or more biological substrates, and which are potentially useful fortriggering biological responses in the tissue by degradation of matrixmolecules, or to activate or release other bioactive compounds in theimplant coating), including Abzymes (antibodies with enzymaticcapacity); adenylate cyclase; alkaline phosphatase; carboxylases;collagenases; cyclooxygenase; hydrolases; isomerases; ligases; lyases;metallo-matrix proteases (MMPs); nucleases; oxidoreductases; peptidases;peptide hydrolase; peptidyl transferase; phospholipase; proteases;sucraseisomaltase; TIMPs; and transferases.

Extracellular Matrix Proteins and non-proteins, including ameloblastin;amelin; amelogenins; collagens (I to XII); dentin-sialo-protein (DSP);dentin-sialo-phospho-protein (DSPP); elastins; enamelin; fibrins;fibronectins; keratins (1 to 20); laminins; tuftelin; carbohydrates;chondroitin sulphate; heparan sulphate; heparin sulphate; hyaluronicacid; lipids and fatty acids; and lipopolysaccarides.

Growth Factors and Hormones (molecules that bind to cellular surfacestructures (receptors) and generate a signal in the target cell to starta specific biological process, such as growth, programmed cell death,release of other molecules (e.g. extracellular matrix molecules orsugar), cell differentiation and maturation, and regulation of metabolicrate) such as Activins (Act); Amphiregulin (AR); Angiopoietins (Ang 1 to4); Apo3 (a weak apoptosis inducer also known as TWEAK, DR3, WSL-1,TRAMP or LARD); Betacellulin (BTC); Basic Fibroblast Growth Factor(bFGF, FGF-b); Acidic Fibroblast Growth Factor (aFGF, FGF-a); 4-1 BBLigand; Brain-derived Neurotrophic Factor (BDNF); Breast and Kidneyderived Bolokine (BRAK); Bone Morphogenic Proteins (BMPs); B-LymphocyteChemoattractant/B cell Attracting Chemokine 1 (BLC/BCA-1); CD27L (CD27ligand); CD3OL (CD30 ligand); CD4OL (CD40 ligand); AProliferation-inducing Ligand (APRIL); Cardiotrophin-1 (CT-1); CiliaryNeurotrophic Factor (CNTF); Connective Tissue Growth Factor (CTGF);Cytokines; 6-cysteine Chemokine (OCkine); Epidermal Growth Factors(EGFs); Eotaxin (Eot); Epithelial Cell-derived Neutrophil ActivatingProtein 78 (ENA-78); Erythropoietin (Epo); Fibroblast Growth Factors(FGF 3 to 19); Fractalkine; Glial-derived Neurotrophic Factors (GDNFs);Glucocorticoid-induced TNF Receptor Ligand (GITRL); Granulocyte ColonyStimulating Factor (G-CSF); Granulocyte Macrophage Colony StimulatingFactor (GM-CSF); Granulocyte Chemotactic Proteins (GCPs); Growth Hormone(GH); 1-309; Growth Related Oncogene (GRO); Inhibins (Inh);Interferon-inducible T-cell Alpha Chemoattractant (I-TAC); Fas Ligand(FasL); Heregulins (HRGs); Heparin-Binding Epidermal Growth Factor-LikeGrowth Factor (HB-EGF); fms-like Tyrosine Kinase 3 Ligand (Flt-3L);Hemofiltrate CC Chemokines (HCC-1 to 4); Hepatocyte Growth Factor (HGF);Insulin; Insulin-like Growth Factors (IGF 1 and 2); Interferon-gammaInducible Protein 10 (IP-10); Interleukins (IL 1 to 18);Interferon-gamma (IFN-gamma); Keratinocyte Growth Factor (KGF);Keratinocyte Growth Factor-2 (FGF-10); Leptin (OB); Leukemia InhibitoryFactor (LIF); Lymphotoxin Beta (LT-B); Lymphotactin (LTN);Macrophage-Colony Stimulating Factor (M-CSF); Macrophage-derivedChemokine (MDC); Macrophage Stimulating Protein (MSP); MacrophageInflammatory Proteins (MIPs); Midkine (MK); Monocyte ChemoattractantProteins (MCP-1 to 4); Monokine Induced by IFN-gamma (MIG); MSX 1 ; MSX2; Mullerian Inhibiting Substance (MIS); Myeloid Progenitor InhibitoryFactor 1 (MPIF-1); Nerve Growth Factor (NGF); Neurotrophins (NTs);Neutrophil Activating Peptide 2 (NAP-2); Oncostatin M (OSM);Osteocalcin; OP-1; Osteopontin; OX40 Ligand; Platelet derived GrowthFactors (PDGF aa, ab and bb); Platelet Factor 4 (PF4); Pleiotrophin(PTN); Pulmonary and Activation-regulated Chemokine (PARC); Regulated onActivation, Normal T-cell Expressed and Secreted (RANTES); Sensory andMotor Neuron-derived Factor (SMDF); Small Inducible Cytokine Subfamily AMember 26 (SCYA26); Stem Cell Factor (SCF); Stromal Cell Derived Factor1 (SDF-1); Thymus and Activation-regulated Chemokine (TARC); ThymusExpressed Chemokine (TECK); TNF and ApoL-related Leukocyte-expressedLigand-1 (TALL-1); TNF-related Apoptosis Inducing Ligand (TRAIL);TNF-related Activation Induced Cytokine (TRANCE); Lymphotoxin InducibleExpression and Competes with HSV Glycoprotein D for HVEM T-lymphocytereceptor (LIGHT); Placenta Growth Factor (PIGF); Thrombopoietin (Tpo);Transforming Growth Factors (TGF alpha, TGF beta 1, TGF beta 2); TumorNecrosis Factors (TNF alpha and beta); Vascular Endothelial GrowthFactors (VEGF-A, B, C and D); calcitonins; and steroid compounds such asnaturally occurring sex hormones such as estrogen, progesterone, andtestosterone as well as analogues thereof.

DNA Nucleic Acids, including A-DNA; B-DNA; artificial chromosomescarrying mammalian DNA (YACs); chromosomal DNA; circular DNA; cosmidscarrying mammalian DNA; DNA; Double-stranded DNA (dsDNA); genomic DNA;hemi-methylated DNA; linear DNA; mammalian cDNA (complimentary DNA; DNAcopy of RNA); mammalian DNA; methylated DNA; mitochondrial DNA; phagescarrying mammalian DNA; phagemids carrying mammalian DNA; plasmidscarrying mammalian DNA; plastids carrying mammalian DNA; recombinantDNA; restriction fragments of mammalian DNA; retroposons carryingmammalian DNA; single-stranded DNA (ssDNA); transposons carryingmammalian DNA; T-DNA; viruses carrying mammalian DNA; and Z-DNA.

RNA Nucleic Acids, including Acetylated transfer RNA (activated tRNA,charged tRNA); circular RNA; linear RNA; mammalian heterogeneous nuclearRNA (hnRNA), mammalian messenger RNA (mRNA); mammalian RNA; mammalianribosomal RNA (rRNA); mammalian transport RNA (tRNA); mRNA;polyadenylated RNA; ribosomal RNA (rRNA); recombinant RNA; retroposonscarrying mammalian RNA; ribozymes; transport RNA (tRNA); virusescarrying mammalian RNA; and short inhibitory RNA (siRNA).

Receptors (cell surface biomolecules that bind signals (such as hormoneligands and growth factors, and transmit the signal over the cellmembrane and into the internal machinery of cells) including, the CDclass of receptors CD; EGF receptors; FGF receptors; Fibronectinreceptor (VLA-5); Growth Factor receptor, IGF Binding Proteins (IGFBP 1to 4); Integrins (including VLA 1-4); Laminin receptor; PDGF receptors;Transforming Growth Factor alpha and beta receptors; BMP receptors; Fas;Vascular Endothelial Growth Factor receptor (Flt-1); and Vitronectinreceptor.

Synthetic Biomolecules, such as molecules that are based on, or mimic,naturally occurring biomolecules.

Synthetic DNA, including A-DNA; antisense DNA; B-DNA; complimentary DNA(cDNA); chemically modified DNA; chemically stabilized DNA; DNA; DNAanalogues; DNA oligomers; DNA polymers; DNA-RNA hybrids; double-strandedDNA (dsDNA); hemimethylated DNA; methylated DNA; single-stranded DNA(ssDNA); recombinant DNA; triplex DNA; T-DNA; and Z-DNA.

Synthetic RNA, including antisense RNA; chemically modified RNA;chemically stabilized RNA; heterogeneous nuclear RNA (hnRNA); messengerRNA (mRNA); ribozymes; RNA; RNA analogues; RNA-DNA hybrids; RNAoligomers; RNA polymers; ribosomal RNA (rRNA); transport RNA (tRNA); andshort inhibitory RNA (siRNA).

Synthetic Biopolymers, including cationic and anionic liposomes;cellulose acetate; hyaluronic acid; polylactic acid; polyglycolalginate; polyglycolic acid; poly-prolines; and polysaccharides.

Synthetic Peptides, including decapeptides comprising DOPA and/ordiDOPA; peptides with sequence “Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys”(SEQ ID NO:2); peptides where a Pro is substituted with hydroxyproline;peptides where one or more Pro is substituted with DOPA; peptides whereone or more Pro is substituted with di-DOPA; peptides where one or moreTyr is substituted with DOPA; peptide hormones; peptide sequences basedon the above listed extracted proteins; and peptides comprising an RGD(Arg Gly Asp) motif. Recombinant Proteins, including all recombinantlyprepared peptides and proteins.

Synthetic Enzyme Inhibitors, including metal ions, that block enzymeactivity by binding directly to the enzyme, molecules that mimic thenatural substrate of an enzyme and thus compete with the principlesubstrate, pepstatin; poly-prolines; D-sugars; D-aminocaids; Cyanide;Diisopropyl fluorophosphates (DFP); N-tosyl-1-phenylalaninechloromethylketone (TPCK); Physostigmine; Parathion; and Penicillin.

Vitamins (Synthetic or Extracted), including biotin; calciferol (VitaminD's; vital for bone mineralisation); citrin; folic acid; niacin;nicotinamide; nicotinamide adenine dinucleotide (NAD, NAD+);nicotinamide adenine dinucleotide phosphate (NADP, NADPH); retinoic acid(vitamin A); riboflavin; vitamin B's; vitamin C (vital for collagensynthesis); vitamin E; and vitamin K's.

Other Bioactive Molecules including adenosine di-phosphate (ADP);adenosine monophosphate (AMP); adenosine tri-phosphate (ATP); aminoacids; cyclic AMP (cAMP); 3,4-dihydroxyphenylalanine (DOPA);5′-di(dihydroxyphenyl-L-alanine (diDOPA); diDOPA quinone; DOPA-likeo-diphenols; fatty acids; glucose; hydroxyproline; nucleosides;nucleotides (RNA and DNA bases); prostaglandin; sugars; sphingosine1-phosphate; rapamycin; synthetic sex hormones such as estrogen,progesterone or testosterone analogues, e.g. Tamoxifene; estrogenreceptor modulators (SERMs) such as Raloxifene; bisphosphonates such asalendronate, risendronate and etidronate; statins such as cerivastatin,lovastatin, simvaststin, pravastatin, fluvastatin, atorvastatin andsodium 3,5-dihydroxy-7-[3-(4-fluorophenyl)-1-(methylethyl)-1H-indol-2-yl]-hept-6-enoate,drugs for improving local resistance against invading microbes, localpain control, local inhibition of prostaglandin synthesis; localinflammation regulation, local induction of biomineralisation and localstimulation of tissue growth, antibiotics; cyclooxygenase inhibitors;hormones; inflammation inhibitors; NSAID's (non-steroid antiinflammatoryagents); painkillers; prostaglandin synthesis inhibitors; steroids, andtetracycline (also as biomineralizing agent). [0049] Biologically ActiveIons, including ions which locally stimulate biological processes likeenzyme function, enzyme blocking, cellular uptake of biomolecules,homing of specific cells, biomineralization, apoptosis, cellularsecretion of biomolecules, cellular metabolism and cellular defense,such as calcium; chromium; copper; fluoride; gold; iodide; iron;potassium; magnesium; manganese; selenium; sulphur; stannum (tin);silver; sodium; zinc; nitrate; nitrite; phosphate; chloride; sulphate;carbonate; carboxyl; and oxide.

Marker Biomolecules, (which generate a detectable signal, e.g. by lightemission, enzymatic activity, radioactivity, specific colour, magnetism,X-ray density, specific structure, antigenicity etc., that can bedetected by specific instruments or assays or by microscopy or animaging method like x-ray or nuclear magnetic resonance, for examplewhich could be employed to monitor processes like biocompatibility,formation of tissue, tissue neogenesis, biomineralisation, inflammation,infection, regeneration, repair, tissue homeostasis, tissue breakdown,tissue turnover, release of biomolecules from the implant surface,bioactivity of released biomolecules, uptake and expression of nucleicacids released from the implant surface, and antibiotic capability ofthe implant surface to demonstrate efficacy and safety validation priorto clinical studies, including calcein; alizaran red; tetracyclins;fluorescins; fura; luciferase; alkaline phosphatase; radiolabeledaminoacids or nucleotides (e.g. marked with ³²P, ³³P, ³H, ³⁵S, ¹⁴C,¹²⁵I, ⁵¹Cr, ⁴⁵Ca); radiolabeled peptides and proteins; radiolabeled DNAand RNA; immuno-gold complexes (gold particles with antibodiesattached); immunosilver complexes; immuno-magnetite complexes; GreenFluorescent protein (GFP); Red Fluorescent Protein (E5); biotinylatedproteins and peptides; biotinylated nucleic acids; biotinylatedantibodies; biotinylated carbon-linkers; reporter genes (any gene thatgenerates a signal when expressed); propidium iodide; and diamidinoyellow.

The probe can also be a cell. The cells can be naturally occurring ormodified cells. In some embodiments, the cells can be geneticallymodified to express surface proteins (e.g., surface antigens) havingknown epitopes or having an affinity for a particular biologicalmolecule of interest. Examples of useful cells include blood cells,liver cells, somatic cells, neurons, and stem cells. Other biologicalpolymers can include carbohydrates, cholesterol, lipids, etc.

While biological molecules can be useful as probes in many applications,the probe itself can be a non-biological molecule. In one case, the dyeprobe can be used for selective biomolecule recognition, as generallydescribed herein. Non-biological probes can also include small organicmolecules that mimic the structure of biological ligands, drugcandidates, catalysts, metal ions, lipid molecules, etc. Also, dyes,markers or other indicating agents can be employed as probes in thepresent invention in order to enable an alternative detection pathway. Acombination of dyes can also be used. Dyes can also be used, in anothercase, as a substrate “tag” to encode a particular substrate or aparticular region on a substrate, for post-processing identification ofthe substrate (polymer probe or target).

Surfaces according to the present invention can also immobilize startermolecules for synthetic applications in particular in solid phasesynthesis, e.g. during the in situ formation of oligo- or polymers.Preferably, the oligo- or polymers are biomolecules and comprisepeptides, proteins, oligo- or polysaccharides or oligo- or polynucleicacids. As immobilized initiators, a monomer of these macromolecules canbe used.

Among the several features of the present invention therefore, is theprovision of a polymer brush for selectively interacting withbiomolecules having improved stability when exposed to an aqueousenvironment; the provision of such a brush wherein improved stability inaqueous environments is achieved by the presence of hydrophobic polymerchains on the substrate surface of the brush, forming a hydrophobiclayer of a controlled thickness; the provision of such a brush whereinpolymer chains having a water-soluble or water-dispersible segmenthaving functional groups capable of bonding to a probe are attached tothe hydrophobic polymer chains; the provision of such a brush whereinthe molecular weight and/or density of the hydrophobic polymer chains iscontrolled to optimize bond stability to the substrate surface; and, theprovision of such a brush wherein the density of the water-soluble orwater-dispersible polymer segments is controlled independent of thehydrophobic polymer chain density, and further is controlled to optimizefunctional group accessibility for probe attachment and/or probeaccessibility for the attachment of a molecule of interest.

Further among the features of the present invention is the provision ofa polymer brush for selectively interacting with biomolecules whereinwater-soluble or water-dispersible polymers, associated with thesubstrate surface of the brush, contain functional groups which attachprobes without the need for chemical activation.

Still further among the features of the present invention is theprovision of a sensor for selectively interacting with biomoleculeswherein polymer chains bound to the substrate surface of the sensor havewater-soluble or water-dispersible segments which contain the residue ofa monomer having a probe for binding the biomolecule already attachedthereto.

Still further among the features of the present invention is theprovision of a polymer brush for selectively interacting withbiomolecules wherein a low density of water-soluble or water-dispersiblepolymer segments are directly or indirectly attached to the substratesurface of the brush, in order to optimize functional groupaccessibility for the attachment of large diameter probes and/or probeaccessibility for the attachment of large diameter molecules.

Still further among the features of the present invention is theprovision of process for preparing a polymer brush for selectivelyinteracting with biomolecules, wherein multiple polymer layers arepresent on the substrate surface of the brush; the provision of such aprocess wherein living free radical polymerization is employed to grow afirst polymer layer from the surface; and, the provision of such aprocess wherein, prior to growth of a second polymer layer from thefirst, a portion of the “living” polymer chain ends are deactivated orterminated, such that additional polymer chain growth does not occur, inorder to control the polymer chain density of the second layer.

The present invention is further directed to methods for preparing thepolymer brushes of the present invention. For example, the presentinvention is further directed to a method of preparing a polymer brushfor binding a molecule in an aqueous sample in an assay, wherein themethod comprises forming a hydrophobic layer on a substrate surfacehaving a dry thickness of at least about 50 angstroms, and then forminga hydrophilic layer on said hydrophobic layer.

Devices that comprise polymer surfaces microstamped by the methods ofthe present invention are thus also an aspect of the invention. As willbe apparent to those of ordinary skill in the art, the direct binding ofbiological and other ligands to polymers is important in many areas ofbiotechnology including, for example, production, storage and deliveryof pharmaceutical proteins, purification of proteins by chromatography,design of biosensors and prosthetic devices, and production of supportsfor attached tissue culture. The present methods find use in creatingdevices for adhering cells and other biological molecules into specificand predetermined positions. Accordingly, one example of a device of thepresent invention is a tissue culture plate comprising at least onesurface microstamped by the method of the present invention. Such adevice could be used in a method for culturing cells on a surface or ina medium and also for performing cytometry.

The present invention is also directed to coat materials for their useas implants and medical devices.

The material to be coated may also be any blood-contacting materialconventionally used for the manufacture of renal dialysis membranes,blood storage bags, pacemaker leads or vascular grafts. For example, thematerial to be modified on its surface may be a polyurethane,polydimethylsiloxane, polytetrafluoroethylene, polyvinylchloride,Dacron™ or Silastic™ type polymer, or a composite made therefrom.

The form of the material to be coated may vary within wide limits.Examples are particles, granules, capsules, fibres, tubes, films ormembranes, preferably moldings of all kinds such as ophthalmic moldings,for example intraocular lenses, artificial cornea or in particularcontact lenses.

Another interesting aspect of polymer brushes is their potential foraffecting a variety of different surface properties, ranging fromadhesion to tribology on many different substrates, and the ability oftuning these properties using an external stimulus. This implicatesapplications such as coatings for corrosion protection to high-techapplications such as controlled-release biocoatings.

Polymer brushes are well-suited for the fabrication of nano- ormicropatterned arrays with control over chemical functionality, shape,and feature dimension and interfeature spacing on the micron andnanometer length scales. These characteristics make polymer brushesattractive for a variety of biotechnological applications includingtheir use in molecular recognition, biosensing, protein separation andchromatography, combinatorial chemistry, scaffolds for tissueengineering, and micro- and nanofluidics.

Adhesion

Whether one considers its promotion or inhibition, adhesion is offundamental importance.

Microbial adhesion is a serious complication after the insertion ofbiomaterials implants or devices in the human body and depends on thephysicochemical surface properties of the adhering microorganisms andthe biomaterial. Polymer brushes increase the distance betweenmicroorganisms and a substratum surface by entropic effects, therewithreducing the attractive forces between surface and the microorganisms.

Biosurfaces

Considerable effort has been made to develop biomaterials that possessgood mechanical properties and biocompatibility. However, they sufferfrom a variety of problems, including poor surface attachment of cellsand tissues. The development of new biomaterials that have all of thedesired properties is costly, and current efforts are focused on usingpresently available biomaterials, but with designed surfaces. Bothadhesion and the inhibition of adhesion are important when consideringapplications involving biosurfaces (e.g., artificial implants, cellculture dishes, biosensors). Many surfaces have been functionalized withproteins and cells by physisorption and “grafting to” methodologies.

Poly(vinylidene difluoride) (PVDF) is used as a biomaterial in softtissue applications. Although its material properties are well-suitedfor this application, improved adhesion of proteins and peptides thatpromote integrin-mediated cell attachment is desired. Tissuecompatibility is engineered by creating poly(acrylic acid) polymerbrushes (plasma-induced SIP) on the PVDIF surface and converting theacid-fiznctionalized brush to a fibronectin-coated surface bycarbodümide coupling reactions, and studied by comparative exposure ofthe modified surface.

Polymer brushes have also found use in this arena particularly throughthe use of surface-attached stimuli-responsive polymers to make “smart”bioconjugates using smart polymers and receptor proteins. The use ofexternal stimuli (e.g., pH, electric field, light, temperature,solvency) to effect a change in polymer properties has also been foundto be very useful for controlling adhesion on biosurfaces. The changeusually comes about from a change in conformation which affectshydrophobicity/hydrophilicity and thus the surface energetics of asurface-attached polymer. Many stimuli-responsive polymers are known,and many studies have been made with those based on poly(Nisopropylacrylamide).

Temperature-responsive surfaces can be created from poly(111PAAM)polymer brushes (via electron beam-initiated polymerization) on tissueculture polystyrene substrates and are used to investigate inflammatorycell adhesion behavior. At elevated temperature, human monocyte andmonocyte-derived macrophages are able to adhere, spread, and fuse toform foreign body giant cells (FBGC) on the hydrophobic surface. Celldetachment is accomplished by lowering the temperature of thebrush-coated surface below the LCST Differential macrophage detachment.

Cell Growth Control

Control of cell growth can be accomplished by attaching cells to asurface, allowing them to proliferate and grow, followed by theirdetachment. Cell attachment and proliferation is a facile process,particularly for hydrophobic surfaces, whereas detachment requiressophistication to recover cells without damage. Thermoresponsive polymerbrushes, with their ability to control hydrophobic/hydrophilicproperties, were investigated to determine their efficacy in thisprocess.

Surface-attached polymers (i.e., both “grafting to” and “grafting from)can be used to control cell growth using protein-repellent micropatternsbased on poly(acrylamide)/PEG copolymers, comb polymers, andpolycationic PEG-grafted copolymers.

Another major field of application for polymer brushes, already widelyexplored for SAMs, is molecular recognition in which biocompatible andnon-biofouling PEG or poly(2-methacryloyloxyethylphosphorylcholine)-containing polymer brushes are patterned ontosurfaces by various lithographic techniques. Subsequently, theunpatterned regions may be backfilled with a biomolecule that gives riseto specific interactions with cells or other biomolecules such asproteins and peptides.

Nonfoulinq Biosurfaces

Recently, polymer brush-coated surfaces provide nonfouling properties.Extracellular proteins adsorb strongly on many surfaces throughhydrophobic interactions. This is useful for making biocoatings.

Tribology

The ability to control surface properties at the nanoscale holds greatpromise for polymer brushes. Polyelectrolyte polymer brushes havesuperior lubrication properties; compared to neutral brushes, and todisplay effective friction coefficients less than 0.0006-0.001 at lowsliding velocities (250-500 nm s-1) and at loading pressures of severalatmospheres in aqueous environments.

Surface Coatings

The wettability of a surface is an important property for manyapplications, and is essential for the creation of an adhesive bond whenjoining two substrates together, during application of a coating to asubstrate and during the creation of almost any interface. Whether theresuiting surface is to be hydrophobic or hydrophilic is highlyapplication-dependent. Super hydrophobic surfaces can be created bycontrolling surface morphology using nanostructures and patternedpolymers. The use of grafted polymers has been used to control wettingin many applications. The control of fiber surface hydrophobicity,wetting, and adhesion properties is important in composite formation.Polymer brushes are prepared on cellulose fibers by grafting from ATRPof methyl acrylate.

Surfaces decorated with poly(4-vinyl-N-methylpyridinum) iodidepolyelectrolyte brushes serve as substrates for the preparation ofwelldefined polyelectrolyte multilayers via layer-by-layer deposition.Strong electrostatic forces and low solubility of the surface-boundpolycation/solution-phase polyanion complex result in nonstoichiometricfilm formation and collapse of this newly formed film to thicknessesnear the dry film thickness.

Coatings can be prepared on electrically conductive substrates usingelectrochemical polymerization. The coatings prepared by this processtend to have highly desirable properties such as good adhesion.Moreover, they can be formed on virtually any shaped substrate, andprocessing can be simplified by the elimination of primers. Thickercoatings can be produced by sequentially coupling cathodicelectropolymerization with another polymerization method. In this way,polymer brushes have been produced on electrically conductive surfaces(e.g., steel, copper etc).

Other applications for polymer brushes include coatings that wouldprovide a barrier to prevent corrosive substances from penetrating anddamaging a substrate. they could make new lubricants in industrialsettings.

Responsive Smart Surfaces

Dependent on the polymer architecture the surface properties (forexample surface energy, i.e. wettability/hydrophobicityund,transparency, light absorption, biologic properties like cell adhesionand microbicidal activity etc.) of the substrates can optionally beinfluenced and changed by external stimuli (solvent parameters,temperature, light, electric fileds).

As a result of this ability to change properties, such polymer brushesare sometimes referred to a stimulus responsive, “switchable” or“smart”.

Now, increasing attention is being paid to the development of responsivesmart surfaces that respond to external stimuli, e.g., light,temperature, electricity, pH, and solvent. Photoswitchable functions offilms or surfaces are desirable for many promising applications. It isnoteworthy that for the construction of smart devices, to graftphotoactive molecules or to prepare photoactive coatings on surfaces isan important and useful route to endow smart devices with some uniquephotoresponsive physical properties, such as wettability, friction,biocompatibility, and optical properties.

Stimuli-Responsive and Switchable Surfaces

The use of stimuli-responsive polymer brushes is very useful in thecontrol of adhesion, particularly in biological applications.

Surface morphology and water contact angle are modified by simply bychanging the solvent to which the block copolymer brush was exposed.Polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) brushes were smooth(RMS roughness=0.77 nm; contact angle −74°) when exposed to CHZCIZ, butbecame rougher (RMS=1.79 nm; contact angle=99° after exposure tocyclohexane.

An interesting application of stimuli-responsive polymer brush surfacesuses a mixed brush composed of poly(2-vinylpyridine) and polyisoprene towrite permanent patterns onto a surface that has been patterned viaphotolithography—a process termed “environment-responsive lithography”.Solvent switching provides both the stimulus for creating and erasingthe pattern. UV radiation during the photolithography step crosslinksthe polyisoprene in the mixed brush, and this causes a loss of switchingproperties for the surface in that region. Imaging relies on thecontrast that develops between parts of the surface that have beenirradiated and masked when exposed to solvent.

Further Potential Applications

Separations

The separation of mixtures into their components is an extremelyimportant process that impacts on all branches of chemistry, andespecially on biological areas where the isolation of pure substances iscritical to their use in humans.

Membranes

The attachment of polymer brushes to membranes can impact a variety offluid flow properties. One might envision that appropriatelyfunctionalized membrane surfaces can improve or enhance separation andresolution through selective adsorption of one component in a mixture.Chiral surfaces could be used for resolving enantiomeric mixtures ofmedicinal products.

Another application of polymer brushes involves their use as microvalvesto control flow. This idea of using two closely spaced polymer brushesas a gate to control fluid flow has been explored both theoretically andexperimentally.

Microfluidics

The development of microfluidic devices is a rapidly growing field whichhas important implications for bioanalytical analysis, studyingreactions in microreactors, and understanding fluid mixing under flow.Interest exists in the possibility that, through the use of patternedpolymer brushes in a microfluidics channel, mixing and fluid flow in thedevice can be controlled.

Microelectronics

Photovoltaics

Polymer Brushes can serve as a substrate for the fabrication ofphotovoltaic devices. The suitable polymer serves as an electron holetransporting component, which together with semiconducting nanocrystalsforms a heterojunction photovoltaic diode with high quantum yields (W.T. S. Huck et al. Nano Lett. 2005, 5, 1653-1657)

Electroless Plating

Metalilization of polymeric substrates is of major importance on the wayto flexible electronics. Polymer Brushes offer a possibility for thesite selective metal deposition for the fabrication of flexiblemicroelectronics. (W. T. S. Huck et al. Langmuir 2006, 22, 6730-6733).

Transistor Fabrication

The use of organic materials in electronic devices such as field effecttransistors or light emmiting diodes is an attractive approach doedecrease weight and cost, simplify the production process and increasethe versatility of such devices. The polymeric dielectric layer for suchdevices should be pinhole-free, with controllable thickness andcomposition. Polymer brushes offer these characteristics and it wasshown, that field effect transistors can be fabricated with them (R. H.Grubbs et al. J. Am. Chem. Soc. 2004, 126, 4062-4063)

The following examples illustrate the present invention without limitingits scope.

EXAMPLE 1

A solid PVC film is prepared by casting a 20% solution of PVC granulate(av. mol weight 60000d, Sigma-Aldrich) in THF on an appropriate supportusing a wire bar system (approx. 1 mm layer thickness). After 2 h dryingon air the film is lifted off and reacted in 250 ml of a 25% aqueousNaN₃ solution and n-tetrabutylammonium bromide (c=40 mmol/1) at 80° C.

For purification the film is treated with water in an ultrasonic bath.

IR spectra clearly show an azidation of the surface.

After activation of the PVC substrate a suitable initiator can becovalently bonded at the surface via a copper-catalysed 1,3-dipolaraddition.

EXAMPLE 2

The PVC film as prepared in Example 1 together with 3.6 g of thealkin-initiator are added to 250 ml of a mixture of DMF and water (5:1),heated up to 65° C. and stirred at this temperature for 1 h.

Then a solution of CuSO₄ (30 mg in 5 ml H₂O) and a solution of sodiumascorbate (127 mg in 5 ml H₂O) are added and stirred over night.

The obtained film has to be extracted for 24 h with diethyl ether inorder to obtain a smooth surface.

EXAMPLE 3

Alternatively to the processes as described in Examples 1 and 2 the PVCsubstrate can also be reacted with a thiol-substituted initiator.

In this case the sulfur reacts as a nucleophile and the initiator isbonded at the PVC surface by substitution of the chlorine.

EXAMPLE 4

33.4 g (119.7 mmol) of (7) is exhibited in a mixture of methanol andwater. After addition of 933.8 mg (5.978 mmol) bipiridyl and 53 mg(0.238 mmol) copper(II)bromide the solution is degassed with nitrogen.

343 mg (2.394 mmol) copper(I)bromide and the activated film are added tothe degassed solution. The reaction mixture is agitated for 1 h at roomtemperature.

For completion of the reaction the film is removed from the reactionmixture, washed in an ultrasonic bath and dried.

The film shows a mass increase of 6.3 mg.

The elemental composition of the PVC sample surface is measured withESCA technique. The size of the analyzed area is 100 micrometers indiameters. The depth of the analysis is 5 nanometers.

The results in the table below are averages of the two measurements.

Surface elemental composition (atomic %) of the PVC sample Sample C O NS PVC 66.4 25.3 4.5 4.0

EXAMPLE 5

2 ml (14.0 mmol) of (9) is exhibited in a mixture of methanol and water.After addition of 28 mg (0.178 mmol) bipiridyl and 2 mg (0.008 mmol) ofcopper(II)bromide the solution is degassed with nitrogen.

12 mg (0.081 mmol) copper(I)bromide and the activated film are added tothe degassed solution. The reaction mixture is agitated for 2 h at roomtemperature.

For completion of the reaction the film is removed from the reactionmixture, washed in an ultrasonic bath and dried.

The film shows a mass increase of 4.8 mg.

EXAMPLE 6

4.3 ml (14.0 mmol) of (11) is exhibited in a mixture of methanol andwater. After addition of 28 mg (0.178 mmol) bipiridyl and 2 mg (0.008mmol) of copper(II)bromide the solution is degassed with nitrogen.

12 mg (0.081 mmol) copper(I)bromide and the activated film are added tothe degassed solution. The reaction mixture is agitated for 2 h at roomtemperature.

For completion of the reaction the film is removed from the reactionmixture, washed in an ultrasonic bath and dried.

The film shows a mass increase of 4.8 mg.

EXAMPLE 7

3.39 ml (13.3 mmol) of (13) is exhibited in iso-propanol.

After addition of 52 mg (0.226 mmol) Me₆TREN and 1.2 mg (0.007 mmol) ofcopper(II)chloride the solution is degassed with nitrogen.

9 mg (0.091 mmol) copper(I)chloride and the activated film are added tothe degassed solution. The reaction mixture is agitated for 64 h at 65°C.

For completion of the reaction the film is removed from the reactionmixture, washed in an ultrasonic bath and dried.

EXAMPLE 8

11.6 g (42.8 mmol) of (15) is exhibited in a mixture of methanol andwater.

After addition of 196 mg (1.255 mmol) bipiridyl and 15 mg (0.067 mmol)of copper(II)bromide the solution is degassed with nitrogen.

73 mg (0.506 mmol) copper(I)bromide and the activated film are added tothe degassed solution. The reaction mixture is agitated for 16 h at roomtemperature.

For completion of the reaction the film is removed from the reactionmixture, washed in an ultrasonic bath and dried.

EXAMPLE 9

Labelling of BSA:

50 mg of BSA (bovine serum albumine, Thermo Scientific) were dissolvedin 20 mM phosphate buffer (pH 7.4). To the solution of BSA in phosphatebuffer was added 0.5 eq Tris(2-carboxyethyl)phosphine hydrochloride andthe mixture was incubated at room temperature for 10 min. Afterwards, 6eq of N-(5-Fluoresceinyl)maleimide (F5M, Sigma-Aldrich)) was added andthe solution was shaken for 5 hours at room temperature. The labelledBSA was isolated using centrifugal filter units. The labelled BSA wascentrifuged and washed with PBS buffer, until no absorbance of the F5M(absorbance maximum 492 nm) was detected using UV spectroscopy. Theconcentrated solution containing the labelled BSA was transferred intoan eppendorf tube and stored at −20° C.

EXAMPLE 10

Covalent Immobilization of BSA:

PVC sheet (1 cm²) 1, PVC carrying polymer brushes with PEG (1 cm²) 2 andPVC carrying polymer brushes with PEG-activated ester group (1 cm²) 3was placed into separate eppendorf vials. To each of the vials was added1 mL solution of fluorescently labelled BSA in 100 mM NaHCO₃ buffer pH8.3. The sheets were shaken at room temperature for 3 hours, afterwardsthe foils was gently removed from the vials and washed extensively with100 mM Na-HCO₃, and stored at 4° C. The foils were analyzed usingfluorescent microscopy. No fluorescence was detected on untreated PVCsheet and PVC with PEG-polymer brushes. PVC with grafted PEG-activatedester group exhibited significant fluorescense response.

EXAMPLE 11

Quantification of Immobilized BSA with Bradford Assay

Bradford Assay:

Standard solutions of BSA with concentration from 2 mg/mL to 0 mg/mLwere prepared. Five different samples of PVC film (prepared as describedabove) (1 cm²) were incubated with a solution of (0.5 mg/mL) BSA in 100mM sodium carbonate buffer pH 8.3 at room temperature. Sample 1—PVCfoil, Sample 2—PVC, carrying polymer brushes with betaine, Sample 3—PVC,bearing polymer brushes with PEG, Sample 4—PVC, bearing polymer brusheswith PEG and an activated group. The samples were incubated for fivehours at room temperature. Samples of 50 μL were taken from eachsolution after 0 min, 1 hour, 2 hours, 3 hours and 5 hours. The sampleswere mixed with 1.5 mL of solution containing the Bradford assay (ThermoScientific), the mixture incubated at room temperature for additional 10min and the absorbance was measured at 465 nm. The protein concentrationin solution was determined using the standard curve obtained for BSA.

LEGEND TABLE 1 Protein concentration samples 1-4 Time (hours) Sample 1Sample 2 Sample 3 Sample 4 0 0.49753 0.5 0.5 0.5 1 0.49411 0.495740.49669 0.49697 2 0.48857 0.49574 0.49669 0.49577 3 0.48857 0.495740.49669 0.49577 5 0.48223 0.49574 0.49669 0.49422

1. A method of preparing a modified halogenated polymer surface,comprising the steps of (a) activating the surface by modification witha polymerization initiator by (a₁) reaction of the halogenated polymersurface with sodium azide and subsequent (a₂) 1,3 dipolar cycloadditionwith an alkine-functionalized initiator; or (a₃) reaction of thehalogenated polymer surface with a mercapto-functionalized initiator;and (b) reacting the activated surface obtained in steps (a₁)/(a₂) or(a₃) with polymerizable monomeric units A and/or B.
 2. Method accordingclaim 1, wherein the initiator represents the fragment of apolymerization initiator capable of initiating polymerization ofethylenically unsaturated monomers in the presence of a catalyst whichactivates controlled radical polymerization.
 3. Method according toclaim 1, wherein the polymerizable monomeric units A and B arecopolymerized by atom transfer radical polymerization (ATRP)participating the initiator of the activated surface obtained in steps(a₁)/(a₂) or (a₃).
 4. Method according to claim 3, wherein the initiatorrepresents the fragment of a polymerization initiator capable ofinitiating polymerization of ethylenically unsaturated monomers in thepresence of a catalyst which activates controlled radicalpolymerization.
 5. Method according to claim 1, wherein the initiator isselected from the group consisting of C₁-C₈alkylhalides,C₆-C₁₅-aralkylhalides, C₂-C₈-haloalkyl esters, arene sulphonylchlorides, haloalkanenitriles, α-haloacrylates and halolactones. 6.Method according to claim 1, wherein the polymerizable monomeric units Aand B differ in polarity and contain one or more olefinic double bond.7. Method according to claim 1, wherein the polymerizable monomericunits A and B are selected from styrenes, acrylic acid,C₁-C₄-alkylacrylic acid, amides, anhydrides orand salts of acrylic acidor C₁-C₄-alkylacrylic acid, acrylic acid-C₁-C₂₄-alkyl esters andC₁-C₄-alkylacrylic acid-C₁-C₂₄-alkyl esters.
 8. Method according toclaim 1, wherein the polymerizable monomeric units A and B are selectedfrom the group consisting of 4-aminostyrene,di-C₁-C₄-alkyl-aminostyrene, styrene, acrylic acid, C₁-C₄-alkylacrylicacid, acrylic or C₁-C₄-alkylacrylamides, acrylic or C₁-C₄alkylacrylmono-or -di-C₁-C₄-alkylamides, acrylic orC₁-C₄-alkylacryl-di-C₁-C₄-alkyl-amino-C₂-C₄-alkylamides, acrylic orC₁-C₄-alkylacryl-amino-C₂-C₄alkylamides, anhydrides orate salts ofacrylic acid or C₁-C₄-alkylacrylic acid, acrylic or C₁-C₄-alkylacrylicacid-mono- or -di-C₁-C₄-alkyl-amino-C₂-C₄-alkyl esters, acrylic orC₁-C₄-alkylacrylic acid-hydroxy-C₂-C₄-alkyl esters, acrylic orC₁-C₄-alkylacrylic acid-(C₁-C₄-alkyl)₃silyloxy-C₂-C₄-alkyl esters,acrylic or C₁-C₄-alkylacrylic acid-(C₁-C₄-alkyl)₃silyl-C₂-C₄-alkylesters, acrylic or C₁-C₄-alkylacrylic acid-heterocyclyl-C₂-C₄-alkylesters, C₁-C₂₄-alkoxylated poly-C₂-C₄-alkylene glycol acrylic orC₁-C₄-alkylacrylic acid esters, acrylic acid-C₁-C₂₄-alkyl esters andC₁-C₄-alkylacrylic acid-C₁-C₂₄-alkyl esters.
 9. A modified halogenatedpolymer surface obtained in the method according to claim
 1. 10. Themodified halogenated polymer surface according to claim 9, whichcorresponds to the formula (1) HalPol-[In-A_(x)-B_(y)C_(z)-Z]_(n),wherein A, B, C represent monomer- oligomer or polymer fragments, whichcan be arranged in block or statstically; Z is halogen which ispositioned at the end of each polymer brush as end group derived fromATRP: HalPol represents the halogenated polymer substrate; In representsthe fragment of a polymerization initiator capable of initiatingpolymerization of ethylenically unsaturated monomers in the presence ofa catalyst which activates controlled radical polymerization; xrepresents a numeral greater than one and defines the number ofrepeating units in A; y represents zero or a numeral greater than zeroand defines the number of monomer, oligopolymer or polymer repeatingunits in B; z represents zero or a numeral greater than zero and definesthe number of monomer, oligopolymer or polymer repeating units in C; andn is one or a numeral greater than one which defines the number ofgroups of the partial formula (1a) In-(A_(x)-B_(y)C_(z)-X)—. 11.(canceled)